Composition comprising xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same and uses thereof

ABSTRACT

This invention provides compositions, methods and process of producing extracts from  Xanthoceras sorbifolia . The extract comprises alkaloids, coumarins, saccharides, proteins, polysaccharides, glycosides, saponins, tannins, acid, flavonoids and others. The composition can be used for treating cancer, arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud&#39;s syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder, impotence and premature ejaculation; for preventing cerebral aging; for improving memory, cerebral functions; or for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer&#39;s disease, autism, brain trauma, Parkinson&#39;s disease or other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud&#39;s syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder. This invention provides compounds comprising at least one sugar, a triterpene, such as Sapogenin, and at least one side chains at Carbon 21 and 22, such as Angeloyl groups. The compounds of the present have various pharmaceutical and therapeutic applications.

This application (1) is a Continuation-In-Part application ofInternational Application No. PCT/US04/43465, Filed Dec. 23, 2004, whichis a Continuation-In-Part application of PCT/US04/33359, Filed Oct. 8,2004, which claims benefit of U.S. Ser. No. 60/509,851, Filed Oct. 9,2003 and U.S. Ser. No. 60/532,101, Filed Dec. 23, 2003; and (2) claimsbenefit of U.S. Ser. Nos. 60/617,379, Filed Oct. 8, 2004; 60/613,811,Filed Sep. 27, 2004; and 60/607,858, Filed Sep. 7, 2004; and (3) claimsbenefit of U.S. Ser. No. 10/471,384, Filed Sep. 4, 2003, correspondingto Int'l App'l No. PCT/IB02/04750, Filed Aug. 28, 2002, which claimspriority of U.S. Ser. No. 09/944,805, Filed Aug. 31, 2001, now U.S. Pat.No. 6,616,943, issued Sep. 9, 2003. The contents of these precedingapplications are hereby incorporated in their entireties by referenceinto this application.

Throughout this application, various publications are referenced.Disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention relates to extracts from a plant called Wenguanguo(Xanthoceras Sorbifolia Bunge), their usages and functions, and methodsof their preparation.

BACKGROUND OF THE INVENTION

Wenguanguo is a species of the sapindaceae family. Its scientific nameis Xanthoceras sorbifolia Bunge. Wenguanguo is the common Chinese name;others are Wenguanngguo, Wenguanmu, Wenguanhua, Xilacedeng andxilasendeng. Goldenhorn and Yellowhorn are its common English names.Wenguanguo is grown in Liaoning, Jilin, Hebei, Shandong, Jiangsu, Henan,Shanxi, Shaanxi, Gansu, Ningxia and Inner Mongolia, China. Its seeds,leaves and flowers are edible and its seeds have been used as a folkmedicine to treat enuresis for centuries. Its branches and woods arealso used as a folk medicine.

Chinese patent applications CN 1092991A and CN 1092992A discussed themethods for producing a medicine from Wenguanguo kernel powder forcuring enuresis and enhancing cerebral functions. Chinese patent CN1052636C discussed a method for producing a medicine with ethanolextract from the Wenguanguo kernel powder for curing enuresis andenhancing cerebral functions. Journal of Shenyang University of Pharmacy(2001), 18(1), 53-56 disclosed the n-butanol extract from the wood ofWenguanguo, which has anti-inflammatory effect.

U.S. Patent Application Publication No. 20030096030 discussed theextracts from the husks of Wenguanguo which are Bunkankasaponin A. B. C.D and two sterols for preventing cerebral aging, improving cerebralfunctions and curing enuresis, frequent micturition, urinaryincontinence, dementia, weak intelligence, and increasing the body'sability to resist the activity of glycosuria.

U.S. Patent Application Publication No. 20030082293 disclosed theextracts Bunkankasaponin A. B. C. D, crude fats, crude protein andsugars from the shell of Wenguanguo.

U.S. Pat. No. 6,616,943, issued on Sep. 9, 2003, discussed thecomposition comprising Wenguanguo combined extracts and the methods forpreparing them and uses thereof. The methods for preparing the combinedextract from the husks comprise the following steps: extractingWenguanguo husks with an organic solvent (e.g. ethanol) to form anorganic (e.g. ethanol) extract; removing the organic solvent (e.g.ethanol) from the organic (e.g. ethanol) extract to form aqueousextracts; and drying and sterilizing the aqueous extracts to form thecombined extracts. The combined extracts contain saponins, saccharides,proteins and others. The extracts can be used for producing medicines orhealth foods for preventing cerebral aging, improving memory, improvingcerebral functions and curing enuresis, frequent micturition, urinaryincontinence, dementia, weak intelligence and Alzheimer's disease,autism, brain trauma, Parkinson's disease and other diseases caused bycerebral dysfunction. The medicines or health foods further compriseVitamin B, Vitamin D, K, anti-oxidant, Cordyceps or its extracts, gingkoor its extracts, Echinacea or its extracts, Huperzine A, folic acid,amino acids, creatine, fiber supplement or a combination thereof.

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull33(4) 1387-1394(1985) described a study on the constituent ofXanthoceras sorbifolia Bunge. See Section V. Saponins from the Fruits ofXanthoceras sorbifolia. Four new saponins were isolated from the fruitsof Xanthoceras sorbifolia Bunge. The structures of these saponins arebunkankasaponins A, B, C and D:

-   (1)    22-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin-   (2)    22-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin-   (3)    28-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin-   (4)    28-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull33(3) 1043-1048 (1985) described studies on the constituent ofXanthoceras sorbifolia Bunge. See Section IV. Structures of the MinerProsapogenin. The prosapogenins from the partial hydrilyzate of fruitsaponin of Xanthoceras sorbifolia were examinated, and are characterizedas:

-   16-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl)protoaecigenin-   22-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl)protoaecigenin    3-O-β-D-glucuronopyranoside

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull33(1) 127-134 (1985) describe studies on the constituent of Xanthocerassorbifolia Bunge. See Section IV. Minor Prosapogenins aponins from theFruits of Xanthoceras sorbifolia Bunge. The structure of 3 minorprosapogenins, obtained by acid hydrolysis of the crude saponin faction,were characterized as:

-   21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyltheasapogenol B-   21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyltheasapogenol B-   21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-22-O-acetylprotoaescigenin

Yingjie Chen, Tadahiro Takeda and Yukio Ogihara in Chem. Pharm. Bull33(4) 1387-1394(1985) described a study on the constituent ofXanthoceras sorbifolia Bunge. See Section II. Major Sapogenol andprosapogenin from the Fruits of Xanthoceras sorbifolia.

Laurence Voutquenne, Cecile Kokougan. Catherine Lavaud, Isabelle Pouny,Marc Litaudon. Triterpenoid saponins and Acylated prosapogenins fromHarpullia austrocalcdonica. Phytochemistry 59 (2002) 825-832

Zhong Jaing, Jean-francois Gallard, Marie-Therese Adeline, VincentDumontet, Mai Van Tri, Thierry Sevenet, and Mary Pais Six TriterpennoidSaponins from Maesa laxiflora. J. Nat. Prod. 1999, 62, 873-876

Young Seo, John M. Berger, Jennine Hoch, Kim M Neddermann, IsiaBursuker, Steven W. Mamber and David G. Kingston. A new TriterpeneSaponin from Pittosporum viridiflorum from the Madagascar Rainforest. J.Nat. Prod. 2002, 65, 65-68

Xiu-Wei Yang, Jing Zhao, Xue-Hui Lui, Chao-Mei Ma, Masao Hattori, and LiHe Zhang Anti-HIV-1 Protease Triterpenoid Saponins from the Seeds ofAesculus chinensis. J. Nat. Prod. 1999 62, 1510-1513

Yi Lu, Tatsuya Umeda, Akihito Yagi, Kanzo Sakata, Tirthankar Chaudhuri,D. K. Ganguly, Secion Sarma. Triterpenoid Saponins from the roots of thetea plant (Camellia sinensis var. Assamica). Phytochchemistry 53 (2000)941-946

Sandra Apers, Tess E. De Bruyne, Magda Claeys, Arnold J. Viletinck, LucA. C. Pieters. New acylated triterpenoid saponins from Maesalaceceolata. Phytochemistry 52 (1999) 1121-1131

-   -   Ilaria D'Acquarica, Maria Cristina, Di Giovanni, Francesco        Gasparrini, Domenico Misiti, Claudio D'Arrigo, Nicolina Fagnano,        Decimo Guarnieri, Giovanni lacono, Giuseppe Bifulco and Raffaele        Riccio. Isolation and structure elucidation of four new        triterpenoid estersaponins from fruits of the Pittosporumtobira        AIT.

Tetrahedron 58 (2002) 10127-10136

Cancer cells are defined by two heritable properties: (1) they reproducein defiance of normal restraints on cell division; and (2) they invadeand colonize territories normally reserved for other cells.

Cancers require mutations of many genes to develop, and they areclassified according to the tissue and cell type from which they arise.Cancers arising from epithelial cells are named carcinomas; thosearising from connective tissue or muscle cells are named sarcomas. Inaddition, there are cancers called leukemias, which are derived fromhemopaietic cells; and cancers derived from cells of the nervous system.

Cancers originating from different types of cells are, in general, verydifferent diseases. Each cancer has characteristics that reflect itsorigin. Even when a cancer has metastasized and proliferated out ofcontrol, its origins can be traced back to a single, primary tumor.Therefore it is important to develop drugs against target cells with aspecified character.

Ovarian cancer is the 5th leading cause of cancer death in women and theleading cause of death from gynecologic. In the United States, femaleshave a 1.4 to 2.5% (1 out of 40-60 women) lifelong chance of developingovarian cancer. Older women are at highest risk. More than half of thedeaths from ovarian cancer occur in women between 55 and 74 years of ageand approximately one quarter of ovarian cancer deaths occur in womenbetween 35 and 54 years of age. (Seehttp://www.nim.nih.gov/medlineplus/ency/article/000889.htm).

Ovarian cancer is disproportionately deadly for a number of reasons.First, symptoms are vague and non-specific, so women and theirphysicians frequently attribute them to more common conditions. By thetime the cancer is diagnosed, the tumor has often spread beyond theovaries. Also, ovarian cancers shed malignant cells that frequentlyimplant on the uterus, bladder, bowel, and lining of the bowel wall(omentum). These cells can begin forming new tumor growths before canceris even suspected. Second, because no cost-effective screening test forovarian cancer exists, more than 50 percent of women with ovarian cancerare diagnosed in the advanced stages of the disease.

This invention provides compounds or compositions extracted fromXanthoceras Sorbifolia or synthesized which have substantial potencyagainst ovarian cancer.

SUMMARY OF THE INVENTION

In accordance with these and other objects of the invention, a briefsummary of the present invention is presented. Some simplifications andomission may be made in the following summary, which is intended tohighlight and introduce some aspects of the present invention, but notto limit its scope. Detailed descriptions of a preferred exemplaryembodiment adequate to allow those of ordinary skill in the art to makeand use the invention concepts will follow in later sections.

The invention provides a compound comprising the following structure,with the formula of C₅₇H₈₈O₂₃ and the name of3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene,also known as Xanifolia-Y This compound was isolated from Xanthocerassorbifolia.

This invention provides a compound comprising the following structure,with the formula of C₆₅H₁₀₀O₂₇ and the name of3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene,also known as Xanifolia-Y1.

The above compounds (Y and Y1) have anti-cancer effect. They inhibit thegrowth of human ovarian cancer (FIG. 3, 4).

These compounds (Y and Y1) are two of the active components identifiedfrom extracts of Xanthoceras sorbifolia by methods of FPLC and HPLC asshown in FIG. 5, 6, 7.

The compound Y is purified with procedure as described in thisapplication (FIG. 7A). The purified compound Y shows 10 times higherpotency (IC50=1.5 ug/ml) than the original extract (IC50=25 ug/ml) (FIG.8). The compound Y has a high selectivity toward ovarian cancer (FIG.9).

The compound Y shows inhibitory activity toward the following humancancer cells (eleven human cancer cell lines were tested in this study)with a higher potency toward ovarian carcinoma (comparison of activitiesis presented in FIG. 10 and Table 3.1).

This invention provides the extract of Xanthoceras Sorbifolia againstcancer growth. The cancer includes, but is not limited to ovary cancer,bladder cancer, prostate cancer, leukocytes cancer, and bone cancer.

The compounds can be isolated from the plant called XanthocerasSorbifolia or can be synthesized chemically, or extracted from otherbiological sources.

This invention is related to the use of extracts of Wenguanguo. Extractsfrom husks, leaves, branches or stems, and fruit-stems, roots and barksof the Wenguanguo can be combined and this invention discloses methodsof their preparation.

The extracts contain saponins, saccharides, proteins, glycosides,flavonoids, curmarin extracts, alkaloid extracts, organic acid extracts,tannin and others.

This invention provides the extract of Xanthoceras Sorbifolia forpreventing cerebral aging; for improving memory; for improving cerebralfunctions; for curing enuresis, frequent micturition, urinaryincontinence, dementia, weak intelligence, Alzheimer's disease, braintrauma, or other diseases caused by cerebral dysfunctions.

Wenguanguo extracts may be used for accelerating the growth of bladder,for suppressing deep sleep, for increasing alertness in a sleepingsubject, for modulating the release, breakdown and uptake ofAntidieuretic hormone (ADH) and its receptors, for modulating thesecretion, breakdown and uptake of Adrenocorticotropic hormone (ACTH)and its receptors, for modulating the release, breakdown and uptake of5-hydroxytryptamine and its receptors, for modulating the release,breakdown and uptake of Acetycholine (Ach) and its receptors, formodulating the release, breakdown and uptake of Adrenaline (AD) and itsreceptors, for modulating the release, breakdown and uptake of Dopamine(DA) and its receptors, for modulating the release, breakdown and uptakeof Norepinephrine (NE) and its receptors, for preventing sleepparalysis, for modulating the formation, release, breakdown and activityof neuropeptides and their receptors, for curing cancer, including butnot limited to breast cancer, leukocyte cancer, liver cancer, ovarycancer, bladder cancer, prostate cancer and brain cancer, and forimproving the functions of the lung and the bladder.

This invention provides a compound comprising a sugar, a triterpene orSapogenin, and side chain at Carbon 21 and/or 22 including Angeloylgroups, operatively linked to form a biologically active compound. In anembodiment, the compound comprises one or more sugars.

This invention provides a salt of the above-described compounds.

This invention provides a pharmaceutical composition comprising aneffective amount of the above-described compounds and a pharmaceuticallyacceptable carrier(s).

This invention provides a method for isolating compounds fromXanthoceras Sorbifolia comprising steps of: extracting XanthocerasSorbifolia powder with an appropriate amount of one or more organicsolvents for an appropriate amount of time to form an organic extract;collecting the organic extract; refluxing the organic extract to form asecond extract; removing the organic solvent from the second extract;drying and sterilizing the second extract to form a XanthocerasSorbifolia extract powder; fractionating the extract powder to obtainone or more components of the extract powder; identifying the bioactivecomponents of the extract powder; purifying one or more bioactivecomponents of the extract powder with FPLC to obtain one or morefraction of the bioactive component; and isolating the pure compoundwith preparative HPLC.

This invention provides a compound having a Structure verified by NMRspectral data derived from proton NMR, Carbon NMR, 2D NMR of theHeteronuclear Multiple Quantum Correlation (HMQC), HeteronuclearMultiple Bond Correlation (HMBC), and COSY, and Mass spectral dataderived from WADLI-TOF and ESI-MS.

This invention provides the chemical features of a compound and itsderivatives which are effective against cancer. The compounds orcompositions of the present invention regulate the receptors orcomponents of cell such as G-protein receptor, Fas protein, receptorTyrosine Kinases, Mitogen, mitogen receptor. The compound inhibitscellular pathways include TGF Beta-smad, FGF, TGF-beta and TGF-alphaa,ras-GTPase-MAP kinase, jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt,myc-cell proliferation, etc. The mutation of cancer cell causes thecell-death program to become inactive, allowing cells to divideindefinitely. The Xanthoceras Sorbifolia derived compound and/orcomposition regulates the components and receptors and re-activates thecell death program.

Abnormal changes in components' activities in pathways cause the cellsto fail to stop proliferating so as to form cancer. The pathways includeTGF Beta-smad, FGF, TGF-beta and TGF-alphaa, ras-GTPase-MAP kinase,jun-fos, Src-fyn, Jak-Jnk-STAT, BMP, Wnt, myc-cell proliferation, etc.The mutation of cancer cell causes the cell-death program to becomeinactive, allowing cells to divide indefinitely. The XanthocerasSorbifolia derived compound and/or composition regulates the componentsand receptors and re-activates the cell death program.

In accordance with these and other objects of the invention, a briefsummary of the present invention is presented. Some simplifications andomission may be made in the following summary, which is intended tohighlight and introduce some aspects of the present invention, but notto limit its scope. Detailed descriptions of a preferred exemplaryembodiment adequate to allow those of ordinary skill in the art to makeand use the inventive concepts will follow in later sections.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure Compound Y with the formula of C₅₇H₈₈O₂₃ andthe chemical name of3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene.

FIG. 2 shows the structure of Compound Y1 with the formula of C₆₅H₁₀₀O₂₇and the chemical name of3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene.

FIG. 3 shows the anticancer activity of purified Compound Y. Theperiment was performed on ovarian cancer cells (OCAR-3) and theinhibition activity was determined by MTT assay (for details, refer toExperiment 3). Abscissa: Concentration (ug/ml). Ordinate: % Cell Growth.The IC50 is approximately 1 ug/ml. A: Point scale. B: Linear scale.

FIG. 4 shows the inhibition of the purified Compound Y1 and Compound Y2on Ovarian cancer cells' growth.

FIG. 5 shows the results of the screening of cell growth activity offractions obtained after FPLC chromatography. The assay was conductedwith bladder cells. The fractions obtained from FPLC as shown in FIG. 20were used. As shown in this figure, that different components ofXanthoceras Sorbifolia extracts cause either growth or inhibitioneffects on cells. Only fraction 5962 (Fraction Y) causes cellinhibition. Fractions 610 and 1116 cause minor stimulation of cellgrowth. Abscissa: concentration (ug/ml). Ordinate: % Cell Growth(determined by MTT assay).

FIG. 6 shows the separation of the components of Xanthoceras Sorbifoliaextract by HPLC with a μbondapak C18 column. Details of experiment waspresented in Experiment 2.

FIG. 7 shows HPLC profile of Fraction Y with 45% Acetonitrile isocraticelution in a preparative C18 column (Delta Pak C18). Under theseconditions, fractions Y, Y1 and Y2 are well separated from each otherand they are collected individually.

FIG. 7A shows the purity of the collected Compound Y by HPLC using 45%acetonitrile isocratic elution in a preparative C18 column.

FIG. 8 shows the a growth curve of ovarian cancer cells after treatmentwith the crude extract of Xanthoceras Sorbifolia as determined by MTTassay. This is a preliminary study on the sensitivity of extract ofXanthoceras Sorbifolia on cancer cell. Cell lines from 11 differenthuman organs were employed. With the crude extract, this figure showsthe most sensitive cancer cells are Ovary cancer cells. Activities onother cancer cells were represented in FIGS. 10A-D.

FIG. 9 shows the comparison of potency of Compound Y between ovariancancer cells and cervical cancer cells. Ovarian cancer cells are muchmore sensitive to Compound Y than the cervical cancer cells. The IC50for Compound Y in ovary cells is about 1.5. This result confirms thatthe activity of compound Y is more selective toward ovary cancer.

FIGS. 10A-D show the growth curves of cancer cells derived fromdifferent human organs as determined by MTT assay. After treatment withthe extract of Xanthoceras Sorbifolia, growth curves of different celllines were presented and their sensitivities (base on IC50 values) weredetermined.

-   -   10A: Sensitive: bladder and bone.    -   10B: Semi-sensitive: leukocyte and liver.    -   10C: Marginal sensitive: prostate, breast and brain.    -   10D: Least sensitive: colon, cervix and lung.

FIG. 11 shows the sprectrum of proton NMR of Compound Y.

FIG. 12 shows 2D NMR (HMQC) results of Compound Y. Also see Table 5.2for the listed chemical shift data.

FIG. 13 shows 2D NMR (HMBC) results of Compound Y. Also see Table 5.3for the listed chemical shift data.

FIG. 14 shows the Mass spectrum of compound Y with MALDI-TOF (highmass): Y+Matrix (CHCA)+Angiotensin 1 “two point calibration”.

FIG. 15 shows the Mass spectrum of compound Y with ESI-MS.

FIG. 16 shows the Proton NMR spectrum of Compound Y1.

FIG. 17 shows the 2D NMR (HMQC) results of Compound Y1. Also see thechemical shift data from Table 6.2.

FIG. 18 shows the 2D NMR (HMBC) results of Y1. Also see the chemicalshift data from Table 6.3.

FIG. 19 shows COSY-NMR profile of Y1 with chemical shift data from Table6.4.

FIG. 20 shows the elution profile of an extract of XanthocerasSorbifolia in FPLC with 10-80% gradient. Ordinate: Optical density (at245 nm). Abscissa: Fractions (5 ml/fraction).

FIG. 21 shows the Proton-NMR spectra of compound R1.

FIG. 22 shows the 2D NMR (HMQC) spectra of compound R1.

FIG. 23 shows the 2D NMR (HMBC) spectra of compound R1.

FIG. 24 shows the 2D NMR (COSY) spectra of compound R1.

FIG. 25 shows the C13 NMR spectra of compound R1.

FIG. 26 shows the chemical structure and the chemical name of CompoundR1.

FIG. 27 shows the chemical structure and the chemical name of CompoundO54.

FIG. 28 shows the Proton-NMR spectra of compound O54.

FIG. 29 shows the 2D NMR (HMQC) spectra of compound O54.

FIG. 30 shows the 2D NMR (HMBC) spectra of compound O54.

FIG. 31 shows one of the four possible chemical structures of Y1. A:structure Y1-1.

FIG. 32 shows one of the four possible chemical structures of Y1. B:structure Y1-2.

FIG. 33 shows one of the four possible chemical structures of Y1. C:structure Y1-3.

FIG. 34 shows one of the four possible chemical structures of Y1. D:structure Y1-4.

FIG. 35 shows the chemical structure of Y-a.

FIG. 36 shows the chemical structure of Y-b.

FIG. 37 shows the chemical structure of Y-c.

FIG. 38 shows the chemical structure of Y1-a.

FIG. 39 shows the chemical structure of Y1-b.

FIG. 40 shows the chemical structure of Y1-c.

FIG. 41 shows the absorption spectrum of Xanthoceras Sorbifolia extract.Abscissa: Wavelength in nm. Ordinate: Optical Density. The extract hasthree absorption maximum at 207 nm, 278 nm and 500 nm.

FIG. 42 shows elution profile of Fraction 5962 with 64% acetonitrileisocratic elution. Two major FPLC fractions X and Y are separated.Ordinate: optical density (254 nm). Abscissa: fraction Number (1ml/fraction).

FIG. 43 shows the comparison of inhibition activity in bladder cells byFractions X (2021) and Y (2728). Only Fraction Y has inhibitionactivity.

FIG. 44 shows HPLC profile of Fraction Y with 45% Acetonitrile isocraticelution. Two major and 2-3 minor compounds were identified.

FIG. 45 shows purification of Fraction R from with FPLC.

FIG. 46 shows the HPLC analysis of fractions #9, #10 and #11 obtainedfrom FPLC.

FIG. 47 shows purification of component-R with HPLC (Delta-Pak C18). A:Extract from fraction #10 of FPLC (iso-30) was further separated byHPLC. B: Rechromatogram of the major component under same condition asdescribed in A.

FIG. 48. Fractionation of Fraction-O from FPLC with HPLC with 20%acetonitrile isocratic elution (iso-20).

FIG. 49. Rechromatography of O28 and O34 (from iso-20).

FIG. 50. Rechromatography of O54 (from iso-20).

FIG. 51 shows the proton NMR spectrum of Y2.

FIG. 52 shows the 2D NMR spectrum of Y2 (HMQC).

FIG. 53 shows the proton NMR spectrum of Y4.

FIG. 54 shows the 2D NMR (HMQC) spectrum of Y4.

FIG. 55 shows the proton NMR spectrum of O28.

FIG. 56 shows the 2D NMR (HMQC).

FIG. 57 shows the proton NMR spectrum of O34.

FIG. 58 shows the 2D NMR (HMQC) spectrum of O34.

FIG. 59 shows the effects of the extract X and Y on the quantity ofurine in mice after 10 days of administration of X and Y.

FIGS. 60(a) and (b) show the water maze learning effect of plant extractadministration of aging mice for 9 days.

FIGS. 61(a) and (b) show the result of water maze learning of 3 daysinjected pentobarbital.

FIG. 62 shows the sleep cycle of a typical person.

FIG. 63. Table 15A-1 shows results of urine volume with water load afteradministration extract for 25 days. FIG. 63A shows the urine volume withwater load after administration of FS(X) and FS(Y) extract for 25 days.

FIG. 64. Table 15A-2 shows results of discharging urine speed with waterload after administration extract for 25 days. FIG. 64A shows thedischarging urine speed with water load after administration of FS(X)and FS(Y) extract for 25 days.

FIG. 65. Table 15A-3 shows results of urine specific gravity and pH withwater load after administration extract for 25 days.

FIG. 66. Table 15A-4 shows concentration of Na+, K+ and Cl− in urinewith water load after administration extract for 25 days.

FIG. 67 shows the structure of the compound.

FIG. 68 shows the structure of the compound.

FIG. 69 shows the structure of the compound.

FIG. 70 shows the structure of the compound.

FIG. 71 shows the structure of the compound.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a compound comprising the following structure,with the formula of C₅₇H₈₈O₂₃ and the name of3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene,also known as Xanifolia-Y This compound was isolated from Xanthocerassorbifolia.

This compound belongs to saponins consist of a triterpene, sugar moietyand angeloyl groups links to the backbone. The angeloyl groups linked tothe C21 and C22 positions. This compound has the anti-cancer activity.

The assignment of this structure is supported by the spectral data (1DH-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS).Accordingly, this compound has the characteristic property as shown inFIGS. 11-15 or Table 5.1.

This invention provides another compound comprising the followingstructure, with the formula of C₆₅H₁₀₀O₂₇ and the name of3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene,also known as Xanifolia-Y1.

This compound belongs to saponins consist of a triterpene, sugar moietyconnected to the backbone. A sugar that linked to the C21 position hastwo angeloyl groups attached. This compound has anti-cancer activity.

The assignment of this structure is supported by the spectral data (1DH-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS).Accordingly, this compound has the characteristic property as shown inFIGS. 16-19 or Table 6.1.

This invention provides evidence to show that the extract of XanthocerasSorbifolia contains anticancer activity. The experiments for determiningthe anti-cancer activity employed human cells lines derived from elevenhuman organs (HTB-9 (bladder), HeLa-S3 (cervix), DU1 45 (prostate), H460(lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2(liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary)). Among the 11cell lines studies, their sensitivity toward Xanthoceras Sorbifoliaextract can be divided into four groups: (A) most sensitive: Ovary (FIG.8); (B) Sensitive: bladder, bone, prostate, and leukocyte, (C) marginalsensitive: liver, breast, and brain; and (D) lease sensitive: colon,cervix, and lung. (FIG. 10A-D). Their IC50 values are listed in Table3.1. TABLE 3.1 IC50 values of Xanthoceras Sorbifolia Extract Determinedin Different Cancer Cells IC50 determined by Cancer cells from differentorgans MTT assay (ug/ml) Ovary (most sensitive) 15-15 Bladder(sensitive) 45-50 Bone 40-55 Prostate 40-50 Leukocyte 45-50 Liver(marginal sensitive) 45-65 Breast  65 Brain 70-85 Colon (leastsensitive)  90 Cervix 115 Lung 110

In order to identify the active compounds of Xanthoceras Sorbifolia, theextract from Xanthoceras Sorbifolia were separated by chromatographycomprising FPLC (Fast Protein Liquid Chromatography) and HPLC (HighPreferment Liquid Chromatography). Multiple fractions were obtained byFPLC procedures (FIG. 20) and HPLC (FIG. 6). Analysis of the componentsof Xanthoceras Sorbifolia by HPLC shows that the extract comprises 26identifiable fractions (named a to z) as shown in FIG. 6.

Anti-cancer activities of these fractions were determined by the MTTassay. Only fraction Ys has the anti-cancer activity (FIG. 5). FractionYs were further separated into 4 components (FIG. 7). The compounds Yand Y1 are the active components currently isolated from XanthocerasSorbifolia as shown in FIG. 3-4.

The invention tested the inhibition effects of ovarian cancer cells withthe MTT assay, and the compound Y shows 10 times higher potency(IC50=1.5 ug/ml) (FIG. 3) than the original crude extract as shown inFIG. 8 (IC50=25 ug/ml).

The selectivity of compound Y was tested, and it has been found thatcompound Y has a much higher potency toward ovarian cancer cells ascompared to the cervical cancer cells (FIG. 9).

The compounds Y1 and Y2 have anti-cancer activity as shown in FIG. 4.

This invention provides the detail isolation procedures for the activecompounds of the present invention.

This invention provides the spectral data evidence (1D H-NMR, C-NMR, 2DNMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, ESI-MS) in supporting theassigned structures.

This invention provides a salt of the above-described compounds.

This invention provides a composition comprising the above-describedcompounds and a suitable carrier.

This invention provides a pharmaceutical composition comprising aneffective amount of the above-described compounds and a pharmaceuticallyacceptable carrier.

This invention provides an anti-ovarian cancer agents and compositioncomprising the above-described composition.

This invention provides the compositions against cancer growth. Thecancer includes, but is not limited to bladder cancer, bone cancer, andovary cancer.

This invention provides a composition comprising the above compounds andtheir derivatives for inhibition of tumour growth.

The following methods and materials were used in the examples and/orexperiments described in this application.

Cells. Human cancer cell lines were obtained from American Type CultureCollection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460(lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2(liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary). Cells were grownin culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN(Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI-1640; HCT-116, U2OSin McCoy-5A) supplemented with 10% fetal calf serum, glutamine andantibiotics in a 5% CO₂ humidified incubator at 37° C.

MTT Assay. The procedure for MTT assay followed the method described in(Carmichael et al., 1987) with only minor modifications. Cells wereseeded into a 96-wells plate at concentrations of 10,000/well (HTB-9,HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU1 45, MCF-7, HepG2,U2OS), or 40,000/well (K562), for 24 hours before drug-treatment. Cellswere then exposed to drugs for 48 hours (72 hours for HepG2, U2OS, and96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/ml) was addedto cultures for an hour. The formation of formazan (product of thereduction of tetrazolium by viable cells) was dissolved with DMSO andthe O.D. at 490 nm was measured by an ELISA reader. The MTT level ofcells before drug-treatment was also measured (T0). The % cell-growth (%G) is calculated as:% G=(TD−T0/TC−T0)×100  (1),

-   -   where TC or TD represent O.D. readings of control or        drug-treated cells. When T0>TD, then the cytotoxicity (LC)        expressed as % of the control is calculated as:        % LC=(TD−T0/T0)×100  (2).

In addition to the compounds Y and Y1, other compounds from the extractincluding R1 and O54, were also purified and the structure weredetermined by 1D H-NMR, C13-NMR, 2D NMR (HMQC, HMBC, COSY); MS(MALDI-TOF).

The Structure of Compound R1 shown below and in FIG. 26, has a chemicalformula of C₆₅H₁₀₆O₂₉ and chemical name of

-   3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene,    also known as Xanifolia-R1.

The assignment of this structure is supported by the spectral data (1DH-NMR, C-NMR, 2D NMR (HMBC, HMQC, COSY), and MS (MALDI-TOF, EMS).Accordingly, this compound has the characteristic property as shown inFIGS. 21-25 or Table 8.1.

Compound-O54

This invention provides a compound O54 with formula of C₆₀H₁₀₀O₂₈ andthe structure was determined by 1D NMR, 2D NMR, MS).

The Structure of Compound O54 (also shown in FIG. 27):

The name of Compound O54 is The chemical name of compound-O54 is:

-   3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene

The assignment of this structure is supported by the spectral data (1 DH-NMR, 2D NMR (HMBC, HMQC). Accordingly, this compound has thecharacteristic property as shown in FIGS. 28-30 and table 9.1.

In other embodiments, the structures of the compounds are as follows:

-   -   Structure 1 as shown in FIG. 31    -   Structure 2 as shown in FIG. 32    -   Structure 3 as shown in FIG. 33    -   Structure 4 as shown in FIG. 34    -   Structure Y-a as shown in FIG. 35    -   Structure Y-b as shown in FIG. 36    -   Structure Y-c as shown in FIG. 37    -   Structure Y1-a as shown in FIG. 38    -   Structure Y1-b as shown in FIG. 39    -   Structure Y1-c as shown in FIG. 40

This invention provides a compound comprising a sugar, a triterpene orSapogenin, and a side chain at Carbon 21 and 22 or Angeloyl groups. Inan embodiment, the compound comprises two or more sugars.

This invention provides a salt of the above-described compounds.

This invention provides a composition comprising the above-describedcompounds and a suitable carrier.

This invention provides a pharmaceutical composition comprising aneffective amount of the above-described compounds and a pharmaceuticallyacceptable carrier.

This invention provides an anti-ovarian cancer agents and compositioncomprising the above-described composition.

This invention provides the compositions against cancer growth. Thecancer includes, but is not limited to bladder cancer, bone cancer, andovary cancer.

This invention provides composition comprising the above compounds andtheir derivatives to inhibit tumour growth.

This invention provides composition comprising the above compounds andtheir derivatives to cure human immunodeficiency virus (HIV) or SevereAcute Respiratory Syndrome (SARS) or flux disease or inhibit virusactivities.

This invention provides medicine or health food for improving thesensory stretch receptor in the bladder wall, inhibiting AChE or use asan anti-inflammatory agent.

This invention provides a method for preventing cerebral aging,improving memory, improving cerebral functions and curing enuresis,frequent micturition, urinary incontinence, dementia, weak intelligenceand Alzheimer's disease, autism, brain trauma, Parkinson's disease andother diseases caused by cerebral dysfunctions, and treating arthritis,rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome,angina pectoris, cardiac disorder, coronary heart disease, headache,dizziness, kidney disorder and treating impotence and prematureejaculation.

This invention provide methods for inhibiting tumor cell growth or totreat patients with HIV or SARS, or inhibit virus activities, or forpreventing cerebral aging, improving memory, improving cerebralfunctions and curing enuresis, frequent micturition, urinaryincontinence, dementia, weak intelligence and Alzheimer's disease,autism, brain trauma, Parkinson's disease and other diseases caused bycerebral dysfunctions, and treating arthritis, rheumatism, poorcirculation, arteriosclerosis, Raynaud's syndrome, angina pectoris,cardiac disorder, coronary heart disease, headache, dizziness, kidneydisorder and treating impotence and premature ejaculation comprisingcontacting an amount of the compound is a triterpene or sapongenin withany two of angeloyl group or tigloyl group or senecioyl group or theircombinations attach to carbon 21 and 22, or any two of angeloyl group ortigloyl group or senecioyl group or their combinations attached to asugar moiety which bonds to carbon 21 or 22.

Wenguanguo is a species of the sapindaceae family. Its scientific nameis Xanthoceras sorbifolia Bunge. Wenguanguo is the common Chinese name;others are Wenguannguo, Wenguanmu, Wenguanhua, and Xilacedeng. Thisplant can grow up to 8 meters in height. It features odd pinnatelycompound leaf, eraceme with white flowers, capsules with thick and woodyhusks. Wenguanguo is grown in Liaoning, Jilin, Hebei, Shandong, Jiangsu,Henan, Shanxi, Shaanxi, Gansu, Ningxia and Inner Mongolia, China. Itsseeds are edible and have been used as a folk medicine to treat enuresisfor centuries. Its branches and woods are also used as a folk medicine.

This invention is a further description of the extracts from Wenguanguo,their uses and methods for preparation. This invention provides theextracts that can prevent enuresis by improving patients' cerebralfunctions so that patients can be more aware of the signals sent fromthe bladder and wake up from deep sleep. When the bladder is full ofurine, the smooth muscle of the bladder is extended, which produces asignal up to the cerebral cortex and cerebellum through the pelvic nerveand the sacral spinal cord. The response of the cerebral cortex andcerebellum to the signal is to make the bladder sustain contracted butthe sphincter relaxed. The urine is then discharged. When the bladder isfilled with urine via the urethra during sleep, the detrusor stretches,allowing the bladder to expand. As the bladder starts to accumulateurine, it will stimulate the stretch receptors in the bladder that willgenerate signals continually to the brain according to the amount ofurine accumulated in the bladder. When the bladder is full enough withurine, then the intra-vesicle has accumulated enough pressure for thebrain to recognize and wake the person to urinate. If the signal is notstrong enough to wake the sleeping person or blocked due to impairmentof cerebral function, then enuresis occurs. This particular plantextract can cure enuresis by improving cerebral functions.

The sensory stretch receptors are located within the bladder wall andhelp with assessing the degree of bladder fullness. This information istransmitted up to the spinal cord and then via the spinothalamic tractsto the central nervous system. The extracts of Wenguanguo make thecentral nervous system more aware of the signal.

When the bladder becomes contracted under stress and nervousness, thecapacity of the urinary bladder will be reduced and then the frequentmicturition occurs. The extracts of Wenguanguo can relax the bladder forstoring more urine.

The capacity of the urinary bladder is reduced because of aging, andthis may even happen to middle-aged people. They suffer from experienceof early detrusor contraction due to a sense of urgency to empty thebladder at low urine level. The extracts of Wenguanguo can help relaxthe detrusor and therefore the bladder capacity increases and urinaryfrequency decreases.

Patients with detrusor overactivity, detrusor instability, detrusorhyper-reflexia or uninhibited bladder have early, forceful detrusorcontractions before the bladder is full. This creates urgency andfrequency urinary discharge. The extract of Wenguanguo relaxes thepatient's detrusor. The bladder becomes stable and can store a fullamount of urine.

The smooth muscle of the urinary bladder has two functions: When thebladder is relaxed, the urine is stored. When it is contracted, theurine will be discharged. The sensory stretch receptors are locatedwithin the bladder wall to assess the bladder's fullness. Thisinformation is transmitted up the spinal cord via the spinothalamictracts to the nervous system. The brain generates inhibitory signalswhen detrusor relaxation is desired. But the brain generates excitatorysignal when detrusor contraction is desired. The extracts of Wenguanguocan relax the bladder tissue by inhibiting Acetylcholinesterase, AchE.The inhibiting effect can be maintained for a long period of time. Theextracts of Wenguanguo are a good AChE inhibitor that can cure thediseases caused by deficiency of Acetylcholine, ACh.

Antidiuretic hormone (ADH) is stored in the posterior pituitary gland inthe brain. It is the primary regulator of body water. ADH acts on thekidneys to increase or decrease total body water. This has an effect onthe volume of urine generated by the kidney. The release of ADH iscontrolled by the cells of osmoreceptors and baroreceptors.Osmoreceptors are the specialized cell hypothalamus. These cells sensethe concentration of particles in the blood. When the concentration ofparticles is higher, more ADH will be released by the pituitary. Thisstimulates retention of water to dilute body fluids. When theconcentration is lower, less ADH will be released by the pituitary.Baroreceptors are located in the right atria and great veins and carotidsinus the specialized area in the heart that sense blood volume andblood pressure. The heart will generate signals to the hypothalamus andpituitary to release more ADH when blood volume or blood pressure is lowand vice versa. The extracts of Wenguanguo can regulate the release ofADH which will reduce the volume of urine produced by the body.

This invention relates to the flavone extracts from Wenguanguo husks andfruit-stems, and methods of their preparation. The methods for preparingthe extracts from Wenguanguo husks and fruit-stems comprise thefollowing steps: extracting Wenguanguo powder made from husk andfruit-stem with ethanol 3-4 times to form an ethanol extract; removingthe ethanol from the ethanol extract to form an aqueous extracts; dryingthe aqueous extracts to form the flavone extracts that is yellow powder.

This invention provides a composition comprising extracts from husks andfruit-stems which are flavonols, flavanols, dihydroflavonols,phenoloids, and others.

This invention relates to the crude flavone extracts from Wenguanguoleaves that includes a water-soluble flavone extracts and awater-insoluble flavone extract and methods of their preparation. Themethods for preparing the extracts from Wenguanguo leaves comprise thefollowing steps: extracting Wenguanguo powder made from the leaves withethanol for 3 times to form an ethanol extract; concentrating theethanol extract to form a concentrated condensed extracts; extractingthe concentrated extract with hot water to from an aqueous extracts anda water-insoluble extract; drying the aqueous extracts and thewater-insoluble extract to form a water-soluble flavone extracts and awater-insoluble flavone extract. This invention provides a compositioncomprising the crude extracts from leaf which are flavonols, flavanols,dihydroflavonols, phenoloids and others.

This invention relates to the flavone extracts from Wenguanguo branchesor stems and methods of their preparation. The methods for preparing theextract from branches or stem comprise the following steps: extractingWenguanguo powder made from the branches or stems with ethanol for 4times to form an ethanol extract; removing the ethanol from the ethanolextract to form an aqueous extracts; drying the aqueous extracts to formflavone extracts which is a yellowish powder.

This invention provides a composition comprising extracts fromWenguanguo branches and stems which are flavonols, flavanols,dihydroflavonols, phenoloids and others.

This invention relates to the flavone extracts from Wenguanguo kernelsand methods of their preparation. The methods for preparing the extractfrom kernels comprise the following steps: removing oil by pressing thekernels to form kernel cakes; grinding and extracting the kernel cakeswith n-hexane to from n-hexane extract; removing the n-hexane from then-hexane extract and drying them to form the kernel powder; extractingthe kernel powder with ethanol to form an ethanol extract; removing theethanol from the ethanol extract to form an aqueous extract; drying theaqueous extracts to form a flavone extracts that is a yellow powder.

This invention provides a composition comprising extracts from kernelwhich are flavonols, flavanols, dihydroflavonols, proteins, phenoloids,and others.

This invention relates to the flavone extract from Wenguanguo root, andmethods of their preparation. The methods for preparing the flavoneextract from Wenguanguo root comprise the following steps: extractingWenguanguo powder made from root with ethanol 3-4 times to form anethanol extract; removing the ethanol from the ethanol extract to forman aqueous extract; drying the aqueous extracts to form the flavoneextracts which is a yellow powder.

This invention provides a composition comprising extracts from roots ofWenguanguo which are flavonols, flavanols, dihydroflavonols, phenoloidsand others.

This invention relates to the flavone extracts from Wenguanguo barks,and methods of their preparation. The methods for preparing the barkextracts from Wenguanguo barks comprise the following steps: extractingWenguanguo powder made from the barks with ethanol 3-4 times to form anethanol extract; removing the ethanol from the ethanol extract to forman aqueous extract; drying the aqueous extracts to form the flavoneextracts which is a yellowish powder.

This invention provides an extract composition from Wenguanguo barkscomprising flavonols, flavanols, dihydroflavonols, phenoloids andothers.

This invention is related to the combined extracts from Wenguanguo husksor fruit-stems and method of their preparation. The methods forpreparing the extract from the husks or fruit-stems comprise thefollowing steps: extracting Wenguanguo powder made from the husks orfruit-stems with an organic solvent (ethanol, methanol and others) toform an organic extract; removing the organic solvent from the organicextract to from an aqueous extracts; drying and sterilizing the aqueousextracts to form the combined extracts.

This invention provides a composition comprising the combined extractsfrom the husks or fruit-stems of the Wenguanguo. The combined extractscomprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguoleaves and method of their preparation. The methods for preparing theextracts from the leaves comprise the following steps: extractingWenguanguo powder made from leaves with an organic solvent (ethanol,methanol and others) to form an organic extract; removing the organicsolvent from the second extract to an aqueous extract; extracting theaqueous extract with ether and water to form an second aqueous extract;extracting the second aqueous extract with n-butanol to form a n-butanolextract; removing the n-butanol from the n-butanol extract to form athird aqueous extract; drying and sterilizing the third aqueous extractto form the combined extracts.

This invention provides a composition comprising the organic extractsfrom the leaves of the Wenguanguo. The organic extracts comprisesaponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguobranches or stems and method of their preparation. The methods forpreparing the extracts from the branches or stems comprise the followingsteps: extracting Wenguanguo powder made from the branches or stems withan organic solvent (ethanol, methanol and others) to form an organicextract; removing the organic solvent from the second extract to anaqueous extract; drying and sterilizing the aqueous extracts to form thecombined extracts.

This invention provides a composition comprising the organic extractsfrom the branches, and stems and of the Wenguanguo. The organic extractscomprise saponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguokernels and method of their preparation. The methods for preparing theextracts from Wenguanguo kernels comprise the following steps: removingoil by pressing the kernels to form kernel cakes; grinding andextracting the kernel cakes with n-hexane to from n-hexane extract;removing the n-hexane from the n-hexane extract and drying them to formthe kernel powder; extracting the kernel powder with an organic solvent(ethanol, methanol and others) to form an organic extract; removing theorganic solvent from the second extract to an aqueous extract; dryingand sterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the organic extractsfrom the kernels of the Wenguanguo. The combined extracts comprisesaponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo rootsand method of their preparation. The methods for preparing the extractsfrom Wenguanguo roots comprise the following steps: extractingWenguanguo powder made from the roots with an organic solvent (ethanol,methanol and others) to form an organic extract; removing the organicsolvent from the organic extract to from an aqueous extracts; drying andsterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the combined extractsfrom the roots of the Wenguanguo. The combined extracts comprisesaponins, saccharides, proteins and others.

This invention is related to the combined extracts from Wenguanguo barksand method of their preparation. The methods for preparing the extractsfrom the barks of Wenguanguo comprise the following steps: extractingWenguanguo powder made from the barks with an organic solvent (ethanol,methanol and others) to form an organic extract; removing the organicsolvent from the organic extract to from an aqueous extract; drying andsterilizing the aqueous extracts to form the combined extracts.

This invention provides a composition comprising the combined extractsfrom the barks of the Wenguanguo. The combined extracts comprisesaponins, saccharides, proteins and others.

This invention provides the crude saponins from the husks or fruit-stemsor seed's shell of Wenguanguo. The methods for preparing the crudesaponins from Wenguanguo husks or fruit-stems comprise the followingsteps: extracting Wenguanguo powder of the husks or fruit-stems with anorganic solvent (ethanol, methanol and others) at ratio of 1:2 for 4-5times, 20-35 hours for each time to form an organic extract; collect andreflux the organic extract 2-3 times at 80° C. to form second extracts;resolve the second extracts in water to form an aqueous solution;extract the aqueous solution by n-butanol to form a n-butanol extracts;chromatograph the n-butanol extracts to form the crude saponins. Thecrude extract comprises saponins.

This invention provides the crude saponins from the leaves of Wenguanguoand method for their preparation. The methods for preparing the crudesaponins from the leaves comprise the following steps: extractingWenguanguo powder of the leaves with an organic solvent (ethanol,methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each timeto form an organic extract; collect and reflux the organic extract 2-3times at 80° C. to form a second extract; resolve the second extracts inwater to form an aqueous solution; extract the aqueous solution byn-butanol to form a n-butanol extracts; chromatograph the n-butanolextracts to form the crude saponins. The crude extract comprisessaponins.

This invention provides the crude saponins from the branches and stemsof Wenguanguo. The methods for preparing the crude saponins from thebranches or stems comprise the following steps: extracting Wenguanguopowder of the branches or stems with an organic solvent (ethanol,methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each timeto form an organic extract; collect and reflux the organic extract 2-3times at 80° C. to form second extracts; resolve the second extracts inwater to form an aqueous solution; extract the aqueous solution byn-butanol to form a n-butanol extracts; chromatograph the n-butanolextracts to form the crude saponins. The crude extract comprisessaponins.

This invention provides the crude saponins from the kernels ofWenguanguo. The methods for preparing the crude saponins from Wenguanguokernels comprise the following steps: removing oil by pressing thekernels to form kernel cakes; grinding and extracting the kernel cakeswith n-hexane to from n-hexane extract; removing the n-hexane from then-hexane extract and dry them to form the kernel powder; extracting thekernel powder with an organic solvent (ethanol, methanol and others) atratio of 1:2, 4-5 times, 20-35 hours each time to form an organicextract; collect and reflux the organic extract for 2-3 times at 80° C.to form second extracts; resolve the second extracts in water to form anaqueous solution; Extract the aqueous solution by n-butanol to form an-butanol extracts; chromatograph the n-butanol extracts to form thecrude saponins. The crude extracts comprise saponins.

This invention provides the crude saponins from the roots of Wenguanguoand method for their preparation. The methods for preparing the crudesaponins from Wenguanguo roots comprise the following steps: extractingWenguanguo powder of the roots with an organic solvent (ethanol,methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each timeto form an organic extract; collect and reflux the organic extract 2-3times at 80° C. to form second extracts; resolve the second extracts inwater to form an aqueous solution; extract the aqueous solution byn-butanol to form a n-butanol extracts; chromatograph the n-butanolextracts to form the crude saponins. The crude extracts containsaponins.

This invention provides the crude saponins from the barks of Wenguanguoand method for their preparation. The methods for preparing the crudesaponins from the barks comprise the following steps: extractingWenguanguo powder of the barks with an organic solvent (ethanol,methanol and others) at a ratio of 1:2, 4-5 times, 20-35 hours each timeto form an organic extract; collect and reflux the organic extract 2-3times at 80° C. to form second extracts; resolve the second extracts inwater to form an aqueous solution; extract the aqueous solution byn-butanol to form a n-butanol extracts; chromatograph the n-butanolextracts to form the crude saponins. The crude extracts comprisesaponins.

This invention provides a process of producing a coumarin extract fromthe husks or fruit-stems of Wenguanguo and their applications. Themethods for preparing the coumarin extracts from husks or fruit-stems ofWenguanguo comprise the following steps: extracting Wenguanguo powder ofthe husks or fruit-stems with 0.5% NaOH solution to form an aqueousextract; collect and extract the aqueous extract by ether to form aether extract; neutralize the ether extract with HCL to form aneutralized ether extract; concentrate and acidize the neutralized etherextract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractsfrom the husks or fruit-stems of Wenguanguo. The extract comprisescoumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract fromthe leaves of Wenguanguo and their applications. The methods forpreparing the coumarin extracts from leaves of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the leaves with 0.5%NaOH solution to form an aqueous extract; collect and extract theaqueous extract by ether to form a ether extract; neutralize the etherextract with HCL to form a neutralized ether extract; concentrate andacidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractsfrom the leaves of Wenguanguo. The extract comprises coumarins, coumaricglycosides and others.

This invention provides a process of producing a coumarin extract fromthe branches and stems of Wenguanguo and their applications. The methodsfor preparing the coumarin extract from the branches or stems ofWenguanguo comprise the following steps: extracting Wenguanguo powderbranches or stems with 0.5% NaOH solution to form an aqueous extract;collect and extract the aqueous extract by ether to form a etherextract; neutralize the ether extract with HCL to form a neutralizedether extract; concentrate and acidize the neutralized ether extract toform the extract comprising crude coumarins.

This invention provides a composition comprising the coumarin extractsfrom the branches and stems of Wenguanguo. The extract comprisescoumarins, coumaric glycosides, saccharides, proteins and others.

This invention provides the crude saponins from the leaves of Wenguanguoand method for their preparation. The methods for preparing the crudesaponins from the leaves comprise the following steps: extractingWenguanguo powder of the leaves with an organic solvent (ethanol,methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each timeto form an organic extract; collect and reflux the organic extract 2-3times at 80° C. to form a second extract; resolve the second extracts inwater to form an aqueous solution; extract the aqueous solution byn-butanol to form a n-butanol extracts; chromatograph the n-butanolextracts to form the crude saponins. The crude extract comprisessaponins.

This invention provides the crude saponins from the branches and stemsof Wenguanguo. The methods for preparing the crude saponins from thebranches or stems comprise the following steps: extracting Wenguanguopowder of the branches or stems with an organic solvent (ethanol,methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each timeto form an organic extract; collect and reflux the organic extract 2-3times at 80° C. to form second extracts; resolve the second extracts inwater to form an aqueous solution; extract the aqueous solution byn-butanol to form a n-butanol extracts; chromatograph the n-butanolextracts to form the crude saponins. The crude extract comprisessaponins.

This invention provides the crude saponins from the kernels ofWenguanguo. The methods for preparing the crude saponins from Wenguanguokernels comprise the following steps: removing oil by pressing thekernels to form kernel cakes; grinding and extracting the kernel cakeswith n-hexane to from n-hexane extract; removing the n-hexane from then-hexane extract and dry them to form the kernel powder; extracting thekernel powder with an organic solvent (ethanol, methanol and others) atratio of 1:2, 4-5 times, 20-35 hours each time to form an organicextract; collect and reflux the organic extract for 2-3 times at 80° C.to form second extracts; resolve the second extracts in water to form anaqueous solution; Extract the aqueous solution by n-butanol to form an-butanol extracts; chromatograph the n-butanol extracts to form thecrude saponins. The crude extracts comprise saponins.

This invention provides the crude saponins from the roots of Wenguanguoand method for their preparation. The methods for preparing the crudesaponins from Wenguanguo roots comprise the following steps: extractingWenguanguo powder of the roots with an organic solvent (ethanol,methanol and others) at ratio of 1:2, 4-5 times, 20-35 hours each timeto form an organic extract; collect and reflux the organic extract 2-3times at 80° C. to form second extracts; resolve the second extracts inwater to form an aqueous solution; extract the aqueous solution byn-butanol to form a n-butanol extracts; chromatograph the n-butanolextracts to form the crude saponins. The crude extracts containsaponins.

This invention provides the crude saponins from the barks of Wenguanguoand method for their preparation. The methods for preparing the crudesaponins from the barks comprise the following steps: extractingWenguanguo powder of the barks with an organic solvent (ethanol,methanol and others) at a ratio of 1:2, 4-5 times, 20-35 hours each timeto form an organic extract; collect and reflux the organic extract 2-3times at 80° C. to form second extracts; resolve the second extracts inwater to form an aqueous solution; extract the aqueous solution byn-butanol to form a n-butanol extracts; chromatograph the n-butanolextracts to form the crude saponins. The crude extracts comprisesaponins.

This invention provides a process of producing a coumarin extract fromthe husks or fruit-stems of Wenguanguo and their applications. Themethods for preparing the coumarin extracts from husks or fruit-stems ofWenguanguo comprise the following steps: extracting Wenguanguo powder ofthe husks or fruit-stems with 0.5% NaOH solution to form an aqueousextract; collect and extract the aqueous extract by ether to form aether extract; neutralize the ether extract with HCL to form aneutralized ether extract; concentrate and acidize the neutralized etherextract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractsfrom the husks or fruit-stems of Wenguanguo. The extract comprisescoumarins, coumaric glycosides and others.

This invention provides a process of producing a coumarin extract fromthe leaves of Wenguanguo and their applications. The methods forpreparing the coumarin extracts from leaves of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the leaves with 0.5%NaOH solution to form an aqueous extract; collect and extract theaqueous extract by ether to form a ether extract; neutralize the etherextract with HCL to form a neutralized ether extract; concentrate andacidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractsfrom the leaves of Wenguanguo. The extract comprises coumarins, coumaricglycosides and others.

This invention provides a process of producing a coumarin extract fromthe branches and stems of Wenguanguo and their applications. The methodsfor preparing the coumarin extract from the branches or stems ofWenguanguo comprise the following steps: extracting Wenguanguo powderbranches or stems with 0.5% NaOH solution to form an aqueous extract;collect and extract the aqueous extract by ether to form a etherextract; neutralize the ether extract with HCL to form a neutralizedether extract; concentrate and acidize the neutralized ether extract toform the extract comprising crude coumarins.

This invention provides a composition comprising the coumarin extractsfrom the branches and stems of Wenguanguo. The extract comprisescoumarins, coumaric glycosides, saccharides, proteins and others.

This invention provides a process of producing a coumarin extract fromthe kernels of Wenguanguo and their applications. The methods forpreparing the coumarin extracts from the kernels of Wenguanguo comprisethe following steps: removing oil by pressing the kernels to form kernelcakes; grinding and extracting the kernel cakes with n-hexane to fromn-hexane extract; removing the n-hexane from the n-hexane extract anddrying them to form the kernel powder; extracting the kernel powder with0.5% NaOH solution to form an aqueous extract; collect and extract theaqueous extract by ether to form an ether extract; neutralizing theether extract with HCL to form a neutralized ether extract; concentrateand acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractsfrom the kernels of Wenguanguo. The extract comprises coumarins,coumaric glycosides and others.

This invention provides a process of producing a coumarin extract fromthe roots of Wenguanguo and their applications. The methods forpreparing the coumarin extract from roots of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the root with 0.5% NaOHsolution to form an aqueous extract; collect and extract the aqueousextract by ether to form a ether extract; neutralize the ether extractwith HCL to form a neutralized ether extract; concentrate and acidizethe neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractsfrom the roots of Wenguanguo. The extract comprises coumarins, coumaricglycosides and others.

This invention provides a process of producing a coumarin extract fromthe barks of Wenguanguo and their applications. The methods forpreparing the coumarin extract from barks of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the bark with 0.5% NaOHsolution to form an aqueous extract; collect and extract the aqueousextract by ether to form a ether extract; neutralize the ether extractwith HCL to form a neutralized ether extract; concentrate and acidizethe neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractfrom the barks of Wenguanguo. The extract comprises coumarins, coumaricglycosides and others.

This invention provides a process of producing an aqueous extract fromthe husks or fruit-stems of Wenguanguo and their applications. Themethod for preparing the water extracts from the husks or fruit-stems ofWenguanguo comprise the following steps: extracting Wenguanguo powder ofthe husk or fruit-stem with water at room temperature for 24 hours toform an aqueous extract; cooking the aqueous extract at 60-70° C. for1-2 hours to form a second water extract; filtering the second waterextract to from a filtered extract; concentrate the filtered extract toform the aqueous extract.

This invention provides a composition comprising the aqueous extractfrom the husks or fruit-stems of Wenguanguo The aqueous extractcomprises sugars, polysaccharides, glycosides, saponins, tannins andothers.

This invention provides a process of producing an aqueous extract fromthe leaves of Wenguanguo and their applications. The method forpreparing the water extracts from the leaves of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the leaves with waterat room temperature for 24 hours to form an aqueous extract; cooking theaqueous extract at 60-70° C. for 1-2 hours to form a second waterextract; filtering the second water extract to from a filtered extract;concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extractfrom leaves of Wenguanguo. The aqueous extract comprises sugars,polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract fromthe branches or stems of Wenguanguo and their applications. The methodfor preparing the water extracts from branches or stems of Wenguanguocomprise the following steps: extracting the Wenguanguo powder of thebranches or stems with water at room temperature for 24 hours to form anaqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hoursto form a second water extract; filtering the second water extract tofrom a filtered extract; concentrating the filtered extract to form theaqueous extract.

This invention provides a composition comprising the aqueous extractfrom the branches or stems of Wenguanguo. The aqueous extract comprisessugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract fromthe kernels of Wenguanguo and their applications. The method forpreparing the water extracts from the kernels of Wenguanguo comprise thefollowing steps: removing oil by pressing the kernels to form kernelcakes; grinding and extracting the kernel cakes with n-hexane to fromn-hexane extract; removing the n-hexane from the n-hexane extract anddry them to form the kernel powder; extracting the kernel powder withwater at room temperature for 24 hours to form an aqueous extract;cooking the aqueous extract at 60-70° C. for 1-2 hours to form a secondwater extract; filtering the second water extract to from a filteredextract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extractfrom kernels of Wenguanguo. The aqueous extract comprises sugars,polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract fromthe roots of Wenguanguo and their applications. The method for preparingthe water extracts from the roots of Wenguanguo comprises the followingsteps: extracting Wenguanguo powder of the roots with water at roomtemperature for 24 hours to form an aqueous extract; cooking the aqueousextract at 60-70° C. for 1-2 hours to form a second water extract;filtering the second water extract to from a filtered extract;concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extractfrom the roots of Wenguanguo The aqueous extract comprises sugars,polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract fromthe barks of Wenguanguo and their applications. The method for preparingthe water extracts from the barks of Wenguanguo comprise the followingsteps: extracting Wenguanguo powder of the barks with water at roomtemperature for 24 hours to form an aqueous extract; cooking the aqueousextract at 60-70° C. for 1-2 hours to form a second water extract;filtering the second water extract to from a filtered extract;concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extractsfrom the barks of Wenguanguo The aqueous extract comprises sugars,polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an alkaloid extract fromthe husks of Wenguanguo and their applications. The methods forpreparing the alkaloid extracts from the husks and fruit-stems ofWenguanguo comprising the following steps: extracting Wenguanguo powderof the husks or fruit-stems with water at a ratio of 1:6, 3-4 times,10-15 hours each time to form an aqueous extract; collect and alkalifythe aqueous extract with NaOH to form a alkalified aqueous extract withpH 10-12; extract the alkalified aqueous extract by toluol to form atoluol extract; the toluol extract flows through 2% of dicarboxylsolution with pH 5-7 to form a dicarboxyl solution; concentrate thedicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extractfrom the husks or fruit-stems of Wenguanguo. The extract comprisesalkaloids and others.

This invention provides a process of producing an alkaloid extract fromthe leaves of Wenguanguo and their applications. The methods forpreparing the alkaloid extract from the leaves of Wenguanguo comprisethe following steps: extracting Wenguanguo powder of the leaves withwater at a ratio of 1:6, 3-4 times, 10-15 hours each time to form anaqueous extract; collecting and alkalifying the aqueous extract withNaOH to form a alkalified aqueous extract with pH 10-12; extracting thealkalified aqueous extract by toluol to form a toluol extract; flow thetoluol extract through 2% of dicarboxyl solution with pH 5-7 to form adicarboxyl solution; concentrate the dicarboxyl solution withdecompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extractfrom the leaves of Wenguanguo. The extract comprises alkaloids andothers.

This invention provides a process of producing an alkaloid extract fromthe branches and stems of Wenguanguo and their applications. The methodsfor preparing the extracts containing alkaloids from branches or stemsof Wenguanguo comprising the following steps: extracting Wenguanguopowder of the branches or stems with water at ratio of 1:6, 3-4 times,10-15 hours each time to form an aqueous extract; collect and alkalifythe aqueous extract with NaOH to form a alkalified aqueous extract withpH 10-12; extracting the alkalified aqueous extract by toluol to form atoluol extract; flow the toluol extract through 2% of dicarboxylsolution with pH 5-7 to form a dicarboxyl solution; concentrate thedicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the extract containingcrude alkaloids from the branches or stems of Wenguanguo. The extractcomprises alkaloids and others.

This invention provides a process of producing an alkaloid extract fromthe kernels of Wenguanguo and their applications. The methods forpreparing the alkaloid extract from kernels of Wenguanguo comprise thefollowing steps: removing oil by pressing the kernels to form kernelcakes; grounding and extracting the kernel cakes with n-hexane to fromn-hexane extract; removing the n-hexane from the n-hexane extract anddry them to form the kernel powder; extracting the kernel powder withwater at ratio of 1:6 for 3-4 times, 10-15 hours for each time to forman aqueous extract; collect and alkalify the aqueous extract with NaOHto form a alkalified aqueous extract with pH 10-12; extract thealkalified aqueous extract by toluol to form a toluol extract; thetoluol extract flows through 2% of dicarboxyl solution with pH 5-7 toform a dicarboxyl solution; concentrate the dicarboxyl solution withdecompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extractfrom the kernels of Wenguanguo. The extract comprises alkaloids andothers.

This invention provides a process of producing an alkaloid extract fromthe roots of Wenguanguo and their applications. The methods forpreparing the alkaloid extract from the roots of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the Wenguanguo rootswith water at a ratio of 1:6, 3-4 times, 10-15 hours each time to forman aqueous extract; collecting and alkalifying the aqueous extract withNaOH to form a alkalified aqueous extract with pH 10-12; extracting thealkalified aqueous extract by toluol to form a toluol extract; flow thetoluol extract through 2% of dicarboxyl solution with pH 5-7 to form adicarboxyl solution; concentrate the dicarboxyl solution withdecompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extractfrom the roots of Wenguanguo. The extract comprises alkaloids andothers.

This invention provides a process of producing an alkaloid extract fromthe barks of Wenguanguo and their applications. The methods forpreparing the alkaloid extract from the barks of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the barks with water atratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueousextract; collect and alkalify the aqueous extract with NaOH to form aalkalified aqueous extract with pH 10-12; extract the alkalified aqueousextract by toluol to form a toluol extract; flow the toluol extractthrough 2% of dicarboxyl solution with pH 5-7 to form a dicarboxylsolution; concentrate the dicarboxyl solution with decompression a toform crude alkaloids.

This invention provides a composition comprising the alkaloid extractfrom the barks of Wenguanguo. The extract comprises alkaloids andothers.

This invention provides a process of producing extract containingorganic acids from husks and fruit-stems and their applications. Themethods for preparing the extracts containing organic acids from thehusks or fruit-stems of Wenguanguo comprise the following steps: extractWenguanguo powder of the husks and or fruit-stems with 10% HCL to forman acid solution; extract the acid solution by an organic solvent (etheror benzol) to form organic extract; extract the organic extract by 5-10%NaHCO3 solution to form a NaHCO₃ extract; acidize and filter the NaHCO3extract to form a deposit matter; extract the deposit matter by anorganic solvent to form the second organic extract; remove the organicsolvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acidsfrom the husks of Wenguanguo. The extract comprising aromatic organicacids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organicacids from leaf and their applications. The methods for preparing theextracts containing organic acids from the leaves of Wenguanguo comprisethe following steps: extract Wenguanguo powder of the leaves with 10%HCL to form an acid solution; extract the acid solution by an organicsolvent (ether or benzol) to form organic extract; extract the organicextract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize andfilter the NaHCO₃ extract to form a deposit matter; extract the depositmatter by an organic solvent to form the second organic extract; removethe organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprisingcrude organic acids extract from the leaves of Wenguanguo. The extractcomprises aromatic organic acids, fatty organic acids, terpenoid organicacids and others.

This invention provides a process of producing extract contains organicacids from branches and stems and their applications. The methods forpreparing the extracts comprising organic acids from the branches orstems of Wenguanguo comprise the following steps: extract Wenguanguopowder of the branches or stems with 10% HCL to form an acid solution;extract the acid solution by an organic solvent (ether or benzol) toform organic extract; extract the organic extract by 5-10% NaHCO₃solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extractto form a deposit matter; extract the deposit matter by an organicsolvent to form the second organic extract; remove the organic solventfrom the second extract to form crude organic acid.

This invention provides a composition comprising the crude organic acidsextract from the branches and stems of Wenguanguo. The extract comprisesaromatic organic acids, fatty organic acids, terpenoid organic acids andothers.

This invention provides a process of producing extract comprise organicacids from kernels and their applications. The methods for preparing theextracts comprising organic acids from the kernels of Wenguanguocomprise the following steps: removing oil by pressing the kernels toform kernel cakes; grounding and extracting the kernel cakes withn-hexane to from n-hexane extract; removing the n-hexane from then-hexane extract and dry them to form the kernel powder; extracting thekernel powder with 10% HCL to form an acid solution; extract the acidsolution by an organic solvent (ether or benzol) to form organicextract; extract the organic extract by 5-10% NaHCO₃ solution to form aNaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a depositmatter; extract the deposit matter by an organic solvent to form thesecond organic extract; remove the organic solvent from the secondextract to form crude organic acid.

This invention provides a composition comprising crude organic acidsextract from the kernels of Wenguanguo. The extract comprises aromaticorganic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organicacids from the roots of Wenguanguo and their applications. The methodsfor preparing the extracts containing organic acids from the roots ofWenguanguo comprise the following steps: extract Wenguanguo powder ofthe roots with 10% HCL to form an acid solution; extract the acidsolution by an organic solvent (ether or benzol) to form organicextract; extract the organic extract by 5-10% NaHCO₃ solution to form aNaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a depositmatter; extract the deposit matter by an organic solvent to form thesecond organic extract; remove the organic solvent from the secondextract to form crude organic acid.

This invention provides a composition comprising the extract comprisingcrude organic acids from the roots of Wenguanguo. The extract comprisesaromatic organic acids, fatty organic acids, terpenoid organic acids andothers.

This invention provides a process of producing extract comprisingorganic acids from barks of Wenguanguo and their applications. Themethods for preparing the extracts containing organic acids from thebarks of Wenguanguo comprise the following steps: extract Wenguanguopowder of the bark with 10% HCL to form an acid solution; extract theacid solution by an organic solvent (ether or benzol) to form organicextract; extract the organic extract by 5-10% NaHCO₃ solution to form aNaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a depositmatter; extract the deposit matter by an organic solvent to form thesecond organic extract; remove the organic solvent from the secondextract to form crude organic acid.

This invention provides a composition comprising the extract comprisingcrude organic acids from the barks of Wenguanguo. The extract comprisesaromatic organic acids, fatty organic acids, terpenoid organic acids andothers.

This invention provides two methods of producing a tannin extract fromWenguanguo husks and fruit-stems and its usage. The first method forpreparing the tannin extract from the husks or fruit-stems of Wenguanguocomprises the following steps: extracting Wenguanguo powder of husks andor fruit-stems with 95% ethanol to form an ethanol extract; concentratethe ethanol extract with decompression a to form the tannin extract. Thesecond method for preparing the tannin extracts from the husks and orfruit-stems of Wenguanguo comprise the following steps: extractingWenguanguo powder of the husks and or fruit-stems with a solvent ofacetone-water at ratio of 1:1 for 2-7 days to form an acetone-waterextract; removing acetone from the acetone-water extract at 50° C. toform a concentrated extract; filtering the concentrated extract to forma filtered extract; extract the filtered extract with ether to form anaqueous extract; extracting the aqueous extract with ethyl acetate andn-butanol to form ethyl acetate and n-butanol extract comprisingtannins.

This invention provides a composition comprising the tannin extractsfrom the husks or fruit-stems of Wenguanguo. The extracts are comprisedof tannins and others.

This invention provides two methods of producing a tannin extract fromWenguanguo leaves and its usage. The first method for preparing thetannin extract from the leaves of Wenguanguo comprise the followingsteps: extracting Wenguanguo powder of the leaves with 95% ethanol toform an ethanol extract; concentrate the ethanol extract withdecompression a to form the tannin extract.

The second method for preparing the tannin extract from the leaves ofWenguanguo comprise the following steps: extracting Wenguanguo powder ofthe leaves with a solvent of acetone-water at ratio of 1:1 for 2-7 daysto form an acetone-water extract; removing acetone from theacetone-water extract at 50° C. to form a concentrated extract;filtering the concentrated extract to form a filtered extract; extractthe filtered extract with ether to form an aqueous extract; extractingthe aqueous extract with ethyl acetate and n-butanol to form ethylacetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract fromthe leaves of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract fromWenguanguo branches and stems and its usage. The first method forpreparing the extracts comprising tannins from branches or stems ofWenguanguo comprise the following steps: extracting Wenguanguo powder ofbranches or stems with 95% ethanol to form an ethanol extract;concentrate the ethanol extract with decompression a to form the tanninextract.

The second method for preparing the tannin extract from the branches orstems of Wenguanguo comprise the following steps: extracting Wenguanguopowder of the branches or stems and with a solvent of acetone-water atratio of 1:1 for 2-7 days to form an acetone-water extract; removingacetone from the acetone-water extract at 50° C. to form a concentratedextract; filtering the concentrated extract to form a filtered extract;extract the filtered extract with ether to form an aqueous extract;extracting the aqueous extract with ethyl acetate and n-butanol to formethyl acetate and n-butanol extract comprising tannins. This inventionprovides a composition comprising the tannin extract from the branch orstem of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract fromWenguanguo kernels and its usage. The first method for preparing thetannin extract from the kernels of Wenguanguo comprise the followingsteps: removing oil by pressing the kernels to form kernel cakes;grinding and extracting the kernel cakes with n-hexane to from n-hexaneextract; removing the n-hexane from the n-hexane extract and dry them toform the kernel powder; extracting the kernel powder with 95% ethanol toform an ethanol extract; concentrating the ethanol extract withdecompression to form the extract comprising tannins.

The second method for preparing the extracts containing tannins from thekernels of Wenguanguo comprise the following steps: removing oil bypressing the kernels to form kernel cakes; grinding and extracting thekernel cakes with n-hexane to from n-hexane extract; removing then-hexane from the n-hexane extract and dry them to form the kernelpowder; extracting the kernel powder with a solvent of acetone-water atratio of 1:1 for 2-7 days to form an acetone-water extract; removingacetone from the acetone-water extract at 50° C. to form a concentratedextract; filtering the concentrated extract to form a filtered extract;extracting the filtered extract with ether to form an aqueous extract;extracting the aqueous extract with ethyl acetate and n-butanol to formethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract fromkernels of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract fromWenguanguo roots and its usage. The first method for preparing thetannin extract from the roots of Wenguanguo comprises the followingsteps: extracting Wenguanguo powder of roots with 95% ethanol to form anethanol extract; concentrating the ethanol extract with decompression toform the tannin extract. The method-2 for preparing the tannin extractfrom the root of Wenguanguo comprises the following steps: extractingWenguanguo powder of the root with a solvent of acetone-water at a ratioof 1:1 for 2-7 days to form an acetone-water extract; removing acetonefrom the acetone-water extract at 50° C. to form a concentrated extract;filtering the concentrated extract to form a filtered extract;extracting the filtered extract with ether to form an aqueous extract;extracting the aqueous extract with ethyl acetate and n-butanol to formethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extractsfrom the roots of Wenguanguo. The extracts comprise tannins and others.

This invention provides two methods of producing tannin extract fromWenguanguo barks and its usage. The method-1 for preparing the tanninextract from the barks of Wenguanguo comprises the following steps:extracting Wenguanguo powder of barks with 95% ethanol to form anethanol extract; concentrating the ethanol extract with decompression toform the tannin extract. The second method for preparing the tanninextract from the barks of Wenguanguo comprising the following steps:extracting Wenguanguo powder of the barks with a solvent ofacetone-water at ratio of 1:1 for 2-7 days to form an acetone-waterextract; removing acetone from the acetone-water extract at 50° C. toform a concentrated extract; filtering the concentrated extract to forma filtered extract; extracting the filtered extract with ether to forman aqueous extract; extracting the aqueous extract with ethyl acetateand n-butanol to form ethyl acetate and n-butanol extract comprisingtannins.

This invention provides a composition comprising the tannin extractsfrom the barks of Wenguanguo. The extracts comprise tannins and others.

This invention provides a method for preventing cerebral aging,improving memory, improving cerebral functions and curing enuresis,frequent micturition, urinary incontinence, dementia, weak intelligenceand Alzheimer's disease, autism, brain trauma, Parkinson's disease andother diseases caused by cerebral dysfunctions, and treating arthritis,rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome,angina pectoris, cardiac disorder, coronary heart disease, headache,dizziness, kidney disorder and treating impotence and prematureejaculation.

According to the theory of traditional Chinese medicine, enuresis,frequent micturition and urinary incontinence are caused by “deficiencyin kidney (“shen”)”. Therefore, they are treated by using Chinese herbswhich can tone the kidney such as Ginseng Bajitian, Roucongrong Duzhongand Cordyceps. These tonifying herbs can strengthen function of thekidney and regulate water metabolism of human's body through the “kidneypathway” that will help with curing the enuresis, frequent micturitionand urinary incontinence.

The Wenguanguo extracts of the present invention can also be used totreat the enuresis, frequent micturition and urinary incontinence.However, the Wenguanguo extracts cure the enuresis, frequent micturitionand urinary incontinence through the “bladder pathway” to regulate watermetabolism of human's body and urination. The Wenguanguo extracts of thepresent invention stimulate the growth of the bladder. See FIG. 10A. TheWenguanguo extracts of the present invention increase the capacity ofbladder and function of bladder controlling the urination. SeeExperiment 15 and Experiment 15A. In another aspect of the presentinvention, Wenguanguo extracts, when used with the “kidney pathway”herbs to treat the enuresis, frequent micturition and urinaryincontinence, will strengthen both the pathways of kidney and bladder,and then will produce better treatment results.

This invention provides the medicines or health foods which furthercomprise Vitamin B, Vitamin D, Vitamin K, grape seed extract and otherantioxidants, Cordyceps or its extract, gingko or its extract, Panaxginseng and P. quinquefolium or their extracts, Huangpi (Clausenalansium) or its extracts, Echinacea or its extract, St John's Wort(Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or itsextract, Tianma (Gastrodia elata) or its extract, Armillariella melleaor its extract, Danshen (Salvia miltiorrhiza) or its extract, Sanqi(Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice),Huanqi (Hedysarum polybotrys) or its extract, D ihuang (Rehmanniaglutinosa) or its extract, Danggui (Angelica sinensis), Yuanzhi(Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or itsextracts, Fuling (Poria cocos) or its extract, enokitake (Flammulinavelutipes) or its extract, Gan Cao (Glycyrrhiza uralensis Fisch) or itsextract, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, aminoacids, creatine, fiber supplement, or any combination thereof.

This invention provides a process of producing a coumarin extract fromthe kernels of Wenguanguo and their applications. The methods forpreparing the coumarin extracts from the kernels of Wenguanguo comprisethe following steps: removing oil by pressing the kernels to form kernelcakes; grinding and extracting the kernel cakes with n-hexane to fromn-hexane extract; removing the n-hexane from the n-hexane extract anddrying them to form the kernel powder; extracting the kernel powder with0.5% NaOH solution to form an aqueous extract; collect and extract theaqueous extract by ether to form an ether extract; neutralizing theether extract with HCL to form a neutralized ether extract; concentrateand acidize the neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractsfrom the kernels of Wenguanguo. The extract comprises coumarins,coumaric glycosides and others.

This invention provides a process of producing a coumarin extract fromthe roots of Wenguanguo and their applications. The methods forpreparing the coumarin extract from roots of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the root with 0.5% NaOHsolution to form an aqueous extract; collect and extract the aqueousextract by ether to form a ether extract; neutralize the ether extractwith HCL to form a neutralized ether extract; concentrate and acidizethe neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractsfrom the roots of Wenguanguo. The extract comprises coumarins, coumaricglycosides and others.

This invention provides a process of producing a coumarin extract fromthe barks of Wenguanguo and their applications. The methods forpreparing the coumarin extract from barks of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the bark with 0.5% NaOHsolution to form an aqueous extract; collect and extract the aqueousextract by ether to form a ether extract; neutralize the ether extractwith HCL to form a neutralized ether extract; concentrate and acidizethe neutralized ether extract to form the coumarin extract.

This invention provides a composition comprising the coumarin extractfrom the barks of Wenguanguo. The extract comprises coumarins, coumaricglycosides and others.

This invention provides a process of producing an aqueous extract fromthe husks or fruit-stems of Wenguanguo and their applications. Themethod for preparing the water extracts from the husks or fruit-stems ofWenguanguo comprise the following steps: extracting Wenguanguo powder ofthe husk or fruit-stem with water at room temperature for 24 hours toform an aqueous extract; cooking the aqueous extract at 60-70° C. for1-2 hours to form a second water extract; filtering the second waterextract to from a filtered extract; concentrate the filtered extract toform the aqueous extract.

This invention provides a composition comprising the aqueous extractfrom the husks or fruit-stems of Wenguanguo The aqueous extractcomprises sugars, polysaccharides, glycosides, saponins, tannins andothers.

This invention provides a process of producing an aqueous extract fromthe leaves of Wenguanguo and their applications. The method forpreparing the water extracts from the leaves of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the leaves with waterat room temperature for 24 hours to form an aqueous extract; cooking theaqueous extract at 60-70° C. for 1-2 hours to form a second waterextract; filtering the second water extract to from a filtered extract;concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extractfrom leaves of Wenguanguo. The aqueous extract comprises sugars,polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract fromthe branches or stems of Wenguanguo and their applications. The methodfor preparing the water extracts from branches or stems of Wenguanguocomprise the following steps: extracting the Wenguanguo powder of thebranches or stems with water at room temperature for 24 hours to form anaqueous extract; cooking the aqueous extract at 60-70° C. for 1-2 hoursto form a second water extract; filtering the second water extract tofrom a filtered extract; concentrating the filtered extract to form theaqueous extract.

This invention provides a composition comprising the aqueous extractfrom the branches or stems of Wenguanguo. The aqueous extract comprisessugars, polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract fromthe kernels of Wenguanguo and their applications. The method forpreparing the water extracts from the kernels of Wenguanguo comprise thefollowing steps: removing oil by pressing the kernels to form kernelcakes; grinding and extracting the kernel cakes with n-hexane to fromn-hexane extract; removing the n-hexane from the n-hexane extract anddry them to form the kernel powder; extracting the kernel powder withwater at room temperature for 24 hours to form an aqueous extract;cooking the aqueous extract at 60-70° C. for 1-2 hours to form a secondwater extract; filtering the second water extract to from a filteredextract; concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extractfrom kernels of Wenguanguo. The aqueous extract comprises sugars,polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract fromthe roots of Wenguanguo and their applications. The method for preparingthe water extracts from the roots of Wenguanguo comprises the followingsteps: extracting Wenguanguo powder of the roots with water at roomtemperature for 24 hours to form an aqueous extract; cooking the aqueousextract at 60-70° C. for 1-2 hours to form a second water extract;filtering the second water extract to from a filtered extract;concentrating the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extractfrom the roots of Wenguanguo The aqueous extract comprises sugars,polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an aqueous extract fromthe barks of Wenguanguo and their applications. The method for preparingthe water extracts from the barks of Wenguanguo comprise the followingsteps: extracting Wenguanguo powder of the barks with water at roomtemperature for 24 hours to form an aqueous extract; cooking the aqueousextract at 60-70° C. for 1-2 hours to form a second water extract;filtering the second water extract to from a filtered extract;concentrate the filtered extract to form the aqueous extract.

This invention provides a composition comprising the aqueous extractsfrom the barks of Wenguanguo The aqueous extract comprises sugars,polysaccharides, glycosides, saponins, tannins and others.

This invention provides a process of producing an alkaloid extract fromthe husks of Wenguanguo and their applications. The methods forpreparing the alkaloid extracts from the husks and fruit-stems ofWenguanguo comprising the following steps: extracting Wenguanguo powderof the husks or fruit-stems with water at a ratio of 1:6, 3-4 times,10-15 hours each time to form an aqueous extract; collect and alkalifythe aqueous extract with NaOH to form a alkalified aqueous extract withpH 10-12; extract the alkalified aqueous extract by toluol to form atoluol extract; the toluol extract flows through 2% of dicarboxylsolution with pH 5-7 to form a dicarboxyl solution; concentrate thedicarboxyl solution with decompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extractfrom the husks or fruit-stems of Wenguanguo. The extract comprisesalkaloids and others.

This invention provides a process of producing an alkaloid extract fromthe leaves of Wenguanguo and their applications. The methods forpreparing the alkaloid extract from the leaves of Wenguanguo comprisethe following steps: extracting Wenguanguo powder of the leaves withwater at a ratio of 1:6, 3-4 times, 10-15 hours each time to form anaqueous extract; collecting and alkalifying the aqueous extract withNaOH to form a alkalified aqueous extract with pH 10-12; extracting thealkalified aqueous extract by toluol to form a toluol extract; flow thetoluol extract through 2% of dicarboxyl solution with pH 5-7 to form adicarboxyl solution; concentrate the dicarboxyl solution withdecompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extractfrom the leaves of Wenguanguo. The extract comprises alkaloids andothers.

This invention provides a process of producing an alkaloid extract fromthe branches and stems of Wenguanguo and their applications. The methodsfor preparing the extracts containing alkaloids from branches or stemsof Wenguanguo comprising the following steps: extracting Wenguanguopowder of the branches or stems with water at ratio of 1:6, 3-4 times,10-15 hours each time to form an aqueous extract; collect and alkalifythe aqueous extract with NaOH to form a alkalified aqueous extract withpH 10-12; extracting the alkalified aqueous extract by toluol to form atoluol extract; flow the toluol extract through 2% of dicarboxylsolution with pH 5-7 to form a dicarboxyl solution; concentrate thedicarboxyl solution with decompression to form the alkaloid extract.

This invention provides a composition comprising the extract containingcrude alkaloids from the branches or stems of Wenguanguo. The extractcomprises alkaloids and others.

This invention provides a process of producing an alkaloid extract fromthe kernels of Wenguanguo and their applications. The methods forpreparing the alkaloid extract from kernels of Wenguanguo comprise thefollowing steps: removing oil by pressing the kernels to form kernelcakes; grounding and extracting the kernel cakes with n-hexane to fromn-hexane extract; removing the n-hexane from the n-hexane extract anddry them to form the kernel powder; extracting the kernel powder withwater at ratio of 1:6 for 3-4 times, 10-15 hours for each time to forman aqueous extract; collect and alkalify the aqueous extract with NaOHto form a alkalified aqueous extract with pH 10-12; extract thealkalified aqueous extract by toluol to form a toluol extract; thetoluol extract flows through 2% of dicarboxyl solution with pH 5-7 toform a dicarboxyl solution; concentrate the dicarboxyl solution withdecompression to form the alkaloid extract.

This invention provides a composition comprising the alkaloid extractfrom the kernels of Wenguanguo. The extract comprises alkaloids andothers.

This invention provides a process of producing an alkaloid extract fromthe roots of Wenguanguo and their applications. The methods forpreparing the alkaloid extract from the roots of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the Wenguanguo rootswith water at a ratio of 1:6, 3-4 times, 10-15 hours each time to forman aqueous extract; collecting and alkalifying the aqueous extract withNaOH to form a alkalified aqueous extract with pH 10-12; extracting thealkalified aqueous extract by toluol to form a toluol extract; flow thetoluol extract through 2% of dicarboxyl solution with pH 5-7 to form adicarboxyl solution; concentrate the dicarboxyl solution withdecompression a to form crude alkaloids.

This invention provides a composition comprising the alkaloid extractfrom the roots of Wenguanguo. The extract comprises alkaloids andothers.

This invention provides a process of producing an alkaloid extract fromthe barks of Wenguanguo and their applications. The methods forpreparing the alkaloid extract from the barks of Wenguanguo comprise thefollowing steps: extracting Wenguanguo powder of the barks with water atratio of 1:6, 3-4 times, 10-15 hours each time to form an aqueousextract; collect and alkalify the aqueous extract with NaOH to form aalkalified aqueous extract with pH 10-12; extract the alkalified aqueousextract by toluol to form a toluol extract; flow the toluol extractthrough 2% of dicarboxyl solution with pH 5-7 to form a dicarboxylsolution; concentrate the dicarboxyl solution with decompression a toform crude alkaloids.

This invention provides a composition comprising the alkaloid extractfrom the barks of Wenguanguo. The extract comprises alkaloids andothers.

This invention provides a process of producing extract containingorganic acids from husks and fruit-stems and their applications. Themethods for preparing the extracts containing organic acids from thehusks or fruit-stems of Wenguanguo comprise the following steps: extractWenguanguo powder of the husks and or fruit-stems with 10% HCL to forman acid solution; extract the acid solution by an organic solvent (etheror benzol) to form organic extract; extract the organic extract by 5-10%NaHCO3 solution to form a NaHCO₃ extract; acidize and filter the NaHCO3extract to form a deposit matter; extract the deposit matter by anorganic solvent to form the second organic extract; remove the organicsolvent from the second extract to form crude organic acid.

This invention provides a composition comprising crude organic acidsfrom the husks of Wenguanguo. The extract comprising aromatic organicacids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organicacids from leaf and their applications. The methods for preparing theextracts containing organic acids from the leaves of Wenguanguo comprisethe following steps: extract Wenguanguo powder of the leaves with 10%HCL to form an acid solution; extract the acid solution by an organicsolvent (ether or benzol) to form organic extract; extract the organicextract by 5-10% NaHCO₃ solution to form a NaHCO₃ extract; acidize andfilter the NaHCO₃ extract to form a deposit matter; extract the depositmatter by an organic solvent to form the second organic extract; removethe organic solvent from the second extract to form crude organic acid.

This invention provides a composition comprising the extract comprisingcrude organic acids extract from the leaves of Wenguanguo. The extractcomprises aromatic organic acids, fatty organic acids, terpenoid organicacids and others.

This invention provides a process of producing extract contains organicacids from branches and stems and their applications. The methods forpreparing the extracts comprising organic acids from the branches orstems of Wenguanguo comprise the following steps: extract Wenguanguopowder of the branches or stems with 10% HCL to form an acid solution;extract the acid solution by an organic solvent (ether or benzol) toform organic extract; extract the organic extract by 5-10% NaHCO₃solution to form a NaHCO₃ extract; acidize and filter the NaHCO₃ extractto form a deposit matter; extract the deposit matter by an organicsolvent to form the second organic extract; remove the organic solventfrom the second extract to form crude organic acid.

This invention provides a composition comprising the crude organic acidsextract from the branches and stems of Wenguanguo. The extract comprisesaromatic organic acids, fatty organic acids, terpenoid organic acids andothers.

This invention provides a process of producing extract comprise organicacids from kernels and their applications. The methods for preparing theextracts comprising organic acids from the kernels of Wenguanguocomprise the following steps: removing oil by pressing the kernels toform kernel cakes; grounding and extracting the kernel cakes withn-hexane to from n-hexane extract; removing the n-hexane from then-hexane extract and dry them to form the kernel powder; extracting thekernel powder with 10% HCL to form an acid solution; extract the acidsolution by an organic solvent (ether or benzol) to form organicextract; extract the organic extract by 5-10% NaHCO₃ solution to form aNaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a depositmatter; extract the deposit matter by an organic solvent to form thesecond organic extract; remove the organic solvent from the secondextract to form crude organic acid.

This invention provides a composition comprising crude organic acidsextract from the kernels of Wenguanguo. The extract comprises aromaticorganic acids, fatty organic acids, terpenoid organic acids and others.

This invention provides a process of producing extract contains organicacids from the roots of Wenguanguo and their applications. The methodsfor preparing the extracts containing organic acids from the roots ofWenguanguo comprise the following steps: extract Wenguanguo powder ofthe roots with 10% HCL to form an acid solution; extract the acidsolution by an organic solvent (ether or benzol) to form organicextract; extract the organic extract by 5-10% NaHCO₃ solution to form aNaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a depositmatter; extract the deposit matter by an organic solvent to form thesecond organic extract; remove the organic solvent from the secondextract to form crude organic acid.

This invention provides a composition comprising the extract comprisingcrude organic acids from the roots of Wenguanguo. The extract comprisesaromatic organic acids, fatty organic acids, terpenoid organic acids andothers.

This invention provides a process of producing extract comprisingorganic acids from barks of Wenguanguo and their applications. Themethods for preparing the extracts containing organic acids from thebarks of Wenguanguo comprise the following steps: extract Wenguanguopowder of the bark with 10% HCL to form an acid solution; extract theacid solution by an organic solvent (ether or benzol) to form organicextract; extract the organic extract by 5-10% NaHCO₃ solution to form aNaHCO₃ extract; acidize and filter the NaHCO₃ extract to form a depositmatter; extract the deposit matter by an organic solvent to form thesecond organic extract; remove the organic solvent from the secondextract to form crude organic acid.

This invention provides a composition comprising the extract comprisingcrude organic acids from the barks of Wenguanguo. The extract comprisesaromatic organic acids, fatty organic acids, terpenoid organic acids andothers.

This invention provides two methods of producing a tannin extract fromWenguanguo husks and fruit-stems and its usage. The first method forpreparing the tannin extract from the husks or fruit-stems of Wenguanguocomprises the following steps: extracting Wenguanguo powder of husks andor fruit-stems with 95% ethanol to form an ethanol extract; concentratethe ethanol extract with decompression a to form the tannin extract. Thesecond method for preparing the tannin extracts from the husks and orfruit-stems of Wenguanguo comprise the following steps: extractingWenguanguo powder of the husks and or fruit-stems with a solvent ofacetone-water at ratio of 1:1 for 2-7 days to form an acetone-waterextract; removing acetone from the acetone-water extract at 50° C. toform a concentrated extract; filtering the concentrated extract to forma filtered extract; extract the filtered extract with ether to form anaqueous extract; extracting the aqueous extract with ethyl acetate andn-butanol to form ethyl acetate and n-butanol extract comprisingtannins.

This invention provides a composition comprising the tannin extractsfrom the husks or fruit-stems of Wenguanguo. The extracts are comprisedof tannins and others.

This invention provides two methods of producing a tannin extract fromWenguanguo leaves and its usage. The first method for preparing thetannin extract from the leaves of Wenguanguo comprise the followingsteps: extracting Wenguanguo powder of the leaves with 95% ethanol toform an ethanol extract; concentrate the ethanol extract withdecompression a to form the tannin extract.

The second method for preparing the tannin extract from the leaves ofWenguanguo comprise the following steps: extracting Wenguanguo powder ofthe leaves with a solvent of acetone-water at ratio of 1:1 for 2-7 daysto form an acetone-water extract; removing acetone from theacetone-water extract at 50° C. to form a concentrated extract;filtering the concentrated extract to form a filtered extract; extractthe filtered extract with ether to form an aqueous extract; extractingthe aqueous extract with ethyl acetate and n-butanol to form ethylacetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract fromthe leaves of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract fromWenguanguo branches and stems and its usage. The first method forpreparing the extracts comprising tannins from branches or stems ofWenguanguo comprise the following steps: extracting Wenguanguo powder ofbranches or stems with 95% ethanol to form an ethanol extract;concentrate the ethanol extract with decompression a to form the tanninextract.

The second method for preparing the tannin extract from the branches orstems of Wenguanguo comprise the following steps: extracting Wenguanguopowder of the branches or stems and with a solvent of acetone-water atratio of 1:1 for 2-7 days to form an acetone-water extract; removingacetone from the acetone-water extract at 50° C. to form a concentratedextract; filtering the concentrated extract to form a filtered extract;extract the filtered extract with ether to form an aqueous extract;extracting the aqueous extract with ethyl acetate and n-butanol to formethyl acetate and n-butanol extract comprising tannins. This inventionprovides a composition comprising the tannin extract from the branch orstem of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract fromWenguanguo kernels and its usage. The first method for preparing thetannin extract from the kernels of Wenguanguo comprise the followingsteps: removing oil by pressing the kernels to form kernel cakes;grinding and extracting the kernel cakes with n-hexane to from n-hexaneextract; removing the n-hexane from the n-hexane extract and dry them toform the kernel powder; extracting the kernel powder with 95% ethanol toform an ethanol extract; concentrating the ethanol extract withdecompression to form the extract comprising tannins.

The second method for preparing the extracts containing tannins from thekernels of Wenguanguo comprise the following steps: removing oil bypressing the kernels to form kernel cakes; grinding and extracting thekernel cakes with n-hexane to from n-hexane extract; removing then-hexane from the n-hexane extract and dry them to form the kernelpowder; extracting the kernel powder with a solvent of acetone-water atratio of 1:1 for 2-7 days to form an acetone-water extract; removingacetone from the acetone-water extract at 50° C. to form a concentratedextract; filtering the concentrated extract to form a filtered extract;extracting the filtered extract with ether to form an aqueous extract;extracting the aqueous extract with ethyl acetate and n-butanol to formethyl acetate and n-butanol extract containing tannins.

This invention provides a composition comprising the tannin extract fromkernels of Wenguanguo. The extract comprises tannins and others.

This invention provides two methods of producing tannin extract fromWenguanguo roots and its usage. The first method for preparing thetannin extract from the roots of Wenguanguo comprises the followingsteps: extracting Wenguanguo powder of roots with 95% ethanol to form anethanol extract; concentrating the ethanol extract with decompression toform the tannin extract. The method-2 for preparing the tannin extractfrom the root of Wenguanguo comprises the following steps: extractingWenguanguo powder of the root with a solvent of acetone-water at a ratioof 1:1 for 2-7 days to form an acetone-water extract; removing acetonefrom the acetone-water extract at 50° C. to form a concentrated extract;filtering the concentrated extract to form a filtered extract;extracting the filtered extract with ether to form an aqueous extract;extracting the aqueous extract with ethyl acetate and n-butanol to formethyl acetate and n-butanol extract comprising tannins.

This invention provides a composition comprising the tannin extractsfrom the roots of Wenguanguo. The extracts comprise tannins and others.

This invention provides two methods of producing tannin extract fromWenguanguo barks and its usage. The method-1 for preparing the tanninextract from the barks of Wenguanguo comprises the following steps:extracting Wenguanguo powder of barks with 95% ethanol to form anethanol extract; concentrating the ethanol extract with decompression toform the tannin extract. The second method for preparing the tanninextract from the barks of Wenguanguo comprising the following steps:extracting Wenguanguo powder of the barks with a solvent ofacetone-water at ratio of 1:1 for 2-7 days to form an acetone-waterextract; removing acetone from the acetone-water extract at 50° C. toform a concentrated extract; filtering the concentrated extract to forma filtered extract; extracting the filtered extract with ether to forman aqueous extract; extracting the aqueous extract with ethyl acetateand n-butanol to form ethyl acetate and n-butanol extract comprisingtannins.

This invention provides a composition comprising the tannin extractsfrom the barks of Wenguanguo. The extracts comprise tannins and others.

This invention provides a method for preventing cerebral aging,improving memory, improving cerebral functions and curing enuresis,frequent micturition, urinary incontinence, dementia, weak intelligenceand Alzheimer's disease, autism, brain trauma, Parkinson's disease andother diseases caused by cerebral dysfunctions, and treating arthritis,rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome,angina pectoris, cardiac disorder, coronary heart disease, headache,dizziness, kidney disorder and treating impotence and prematureejaculation.

According to the theory of traditional Chinese medicine, enuresis,frequent micturition and urinary incontinence are caused by “deficiencyin kidney (“shen”)”. Therefore, they are treated by using Chinese herbswhich can tone the kidney such as Ginseng Bajitian, Roucongrong Duzhongand Cordyceps. These tonifying herbs can strengthen function of thekidney and regulate water metabolism of human's body through the “kidneypathway” that will help with curing the enuresis, frequent micturitionand urinary incontinence.

The Wenguanguo extracts of the present invention can also be used totreat the enuresis, frequent micturition and urinary incontinence.However, the Wenguanguo extracts cure the enuresis, frequent micturitionand urinary incontinence through the “bladder pathway” to regulate watermetabolism of human's body and urination. The Wenguanguo extracts of thepresent invention stimulate the growth of the bladder. See FIG. 10A. TheWenguanguo extracts of the present invention increase the capacity ofbladder and function of bladder controlling the urination. SeeExperiment 15 and Experiment 15A. In another aspect of the presentinvention, Wenguanguo extracts, when used with the “kidney pathway”herbs to treat the enuresis, frequent micturition and urinaryincontinence, will strengthen both the pathways of kidney and bladder,and then will produce better treatment results.

This invention provides the medicines or health foods which furthercomprise Vitamin B, Vitamin D, Vitamin K, grape seed extract and otherantioxidants, Cordyceps or its extract, gingko or its extract, Panaxginseng and P. quinquefolium or their extracts, Huangpi (Clausenalansium) or its extracts, Echinacea or its extract, St John's Wort(Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or itsextract, Tianma (Gastrodia elata) or its extract, Armillariella melleaor its extract, Danshen (Salvia miltiorrhiza) or its extract, Sanqi(Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice),Huanqi (Hedysarum polybotrys) or its extract, D ihuang (Rehmanniaglutinosa) or its extract, Danggui (Angelica sinensis), Yuanzhi(Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or itsextracts, Fuling (Poria cocos) or its extract, enokitake (Flammulinavelutipes) or its extract, Gan Cao (Glycyrrhiza uralensis Fisch) or itsextract, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, aminoacids, creatine, fiber supplement, or any combination thereof.

There are many different periods of sleep a person goes through. Theseinclude Slow-Wave-Sleep 1 (SWS 1), Slow-Wave-Sleep 2 (SWS 2),Slow-Wave-Sleep 3 (SWS 3) Slow-Wave-Sleep 4 (SWS 4) and Rapid EyeMovement (REM). SWS 1 and SWS 2 are both periods of light sleep where itis relatively easy to wake someone up. Light sleep is usually morefrequent in the second half of sleep. SWS 3 and SWS 4 are both periodsof deep sleep, where it is difficult to wake the sleeper. Deep sleep ismore frequent in the first half of sleep and each period will getshorter each time afterward. REM is a period of sleep in which peoplehave their most vivid dreams. The wave patterns are similar to thepatterns in which a person is awake. However, it is difficult to wakesomeone up who is in this state of sleep. The sleep cycle of a typicalperson can be described as follows:

SWS1, SWS2, SWS3, SWS4, SWS3, SWS2, REM, SWS1, SWS2, SWS3, SWS4, SWS3,SWS2, REM. (See FIG. 62).

However, the above sequence may not be in a fixed order. The sleepstatus may shift from SWS 4 to SWS1 or wake up because the body turnsfrom one side to the other. It may shift to the SWS2 status after themovement. The interval between two REM is about 90 min. For healthypeople, SWS1 will occupy about 5% of sleep, SWS2 will occupy about 50%of sleep, SWS3 will occupy about 10% of sleep, SWS4 will occupy about10% of sleep and REM will occupy about 25% of sleep. Since a person inSWS1 and SWS2 can easily be woken, a healthy person has enoughopportunities to wake up to urinate. However if a person's sleepingstatus is mostly in SWS4, he has less chance of waking up when thebladder is full. It is difficult for him to break through the barriersof deep sleep. Then enuresis occurs. This invention relates to a plantextract, including Wenguanguo, for preventing enuresis.

This invention provides the extract of Wenguanguo for inhibiting theuptake of 5-hydroxytryptamine (5HT) in a subject.

5-HT controls and modulates a sleep factor that sustains and increasesdeep sleep. Inhibiting the uptake of 5HT will decrease deep sleep.People who spend too much time in SWS 3 and SWS 4 are unable to awakenfrom their sleep when their bladder is full because their sleep is toodeep. This is the reason that enuresis often occurs during SWS 3 and SWS4.

This invention provides the extract of Wenguanguo for increasing theactivity of Dopamine in a subject thereby making the central nervesystem of said subject alert.

This invention provides the extract of Wenguanguo for increasing thesecretion of antidiuretic hormone (ADH) in a subject, which reducesurine in said subject.

This invention provides the extract of Wenguanguo for modulating therelease, breakdown and uptake of Acetylcholine (Ach) and its receptorsin a subject. The said extracts of this invention inhibits the deepsleep created by 5HT and increase REM sleep.

This invention provides the extract of Wenguanguo for preventing sleepparalysis in a subject.

This invention provides the extract of Wenguanguo for providingalertness to a sleeping subject.

This invention provides the extract for helping the growth of thebladder and sphincter.

An immature bladder and sphincter cannot control the process and actionof urination. By accelerating the growth of the bladder and thesphincter, this problem will be overcome, and enuresis will not occur.

This invention provides the extract of Wenguanguo against cancer growth.The cancer includes, but is not limited to bladder cancer, cervixcancer, prostate cancer, lung cancer, breast cancer, leukocytes cancer,colon cancer, liver cancer, bone cancer, skin cancer, brain cancer, andovary cancer.

This invention provides the extract of Wenguanguo inhibit tumoractivities.

This invention provides the pathways interacted by compounds isolatedfrom Xanthoceras Sorbifolia. In an embodiment, a compound has theformula C₅₇H₈₈O₂₃ and the chemical name3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene,designated herein as “Structure Y”, and derivative compounds which areeffective against cancer. In another embodiment, the compounds of thepresent invention comprise the chemical structures designated herein as“Structure Y1”, “Structure R1”, “Structure 1 to 4”, “Structure Y-a toY-c” and “Structure Y1-a to Y1-c”, “Structure Y1-1 to Y1-4” and theirderivatives. See FIG. 31-40.

They regulate the receptors or components of cells. The compounds can beisolated from the plant called Xanthoceras Sorbifolia or can besynthesized.

The compounds of the present invention have structures as shown below:

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene

-   -   Structure 1 as shown in FIG. 31    -   Structure 2 as shown in FIG. 32    -   Structure 3 as shown in FIG. 33    -   Structure 4 as shown in FIG. 34    -   Structure Y-a as shown in FIG. 35    -   Structure Y-b as shown in FIG. 36    -   Structure Y-c as shown in FIG. 37    -   Structure Y1 (also shown in FIG. 2).

3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene

-   -   Structure Y1-a as shown in FIG. 38    -   Structure Y1-b as shown in FIG. 39    -   Structure Y1-c as shown in FIG. 40.

This invention further provides a compound comprising the followingstructure:

Structure R1:3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene

This invention further provides a compound comprising the followingstructure:

The structure of compound O54 is presented in the following figure.

The chemical name of compound-O54 is:

-   O54:    3-O-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene.

There are many components and pathways monitoring cell proliferation.

The Xanthoceras Sorbifolia compound or its derivatives work in the Wnt(Wingless-type MMTV integration site family member) signaling pathway.The Wnt signaling pathway is evolutionarily conserved and controls manyevents during the embryogenesis. This pathway regulates cell morphology,proliferation, motility and as well as cell apoptosis. It also plays animportant role during tumorigenesis. The Wnt pathway has also beenobserved as inappropriately activated in several different types ofcancers in humans.

In the nucleus, the target genes for Wnt signaling are normally keptsilent by an inhibitory complex of gene regulatory proteins, e.g. theGroucho corepressor protein bound to the gene regulatory proteinLEF-I/TCF. In the absence of a Wnt signal, some β-cartenin is bound tothe cytosolic tail of cadherin proteins, and any cytosolic β-carteninthat becomes bound by the APC-axin-GSK-3β will trigger itsubiquitylation and degradation in proteasomes. The result is thedecrease of intracellular amount of β-cartenin. However, when the Wntbinding to Frizzled (a seven transmembrane receptor) and LRP (Lowdensity lipoprotein Receptor) activates Dishevelled (a cytoplasmicsignaling protein) by a mechanism, this leads to the inactivation ofGSK-β3 in the degradation complex by a mechanism which requires caseinkinase I, as well as casein kinase II. The activity of the multiproteincomplex of β-catenin-axin-adenomatous-polyposis coli (APC)-glycogensynthase kinase (GSK)-3β, which targets β-catenin by phosphorylation fordegradation by the proteasome, is then inhibited by Dsh/Dvl(Dishevelled, dsh homolog 1). This then inhibits priming of β-catenin,and indirectly prevents the GSK-3β phosphorylation of β-catenin. Whenstimulated by Wnt, Dvl recruits the GSK-3 binding protein, GBP, to themultiprotein complex of β-catenin-axin-adenomatous-polyposis coli(APC)-glycogen synthase kinase (GSK)-3β. GBP then titrates GSK-β fromaxin, and in this way, phosphorylation of β-catenin is inhibited. Then,axin is sequestrated by LRP at the cell membrane. The result of all ofthis is an accumulation of cytosolic β-catenin. In the nucleus,β-catenin binds to LEF-I/TCF, displaces Groucho, and acts a co-activatorto stimulate the transcription of Wnt target genes.

Xanthoceras Sorbifolia compositions regulate the components related toWnt pathways or its receptors, thereby stopping the proliferation ofcancer cells.

The compound or its derivatives work in the Mitogens, Ras and a MAP(Mitogen activation protein) kinase pathway. Mitogens stimulate celldivision. The binding of mitogens to cell-surface receptors leads to theactivation of Ras and a MAP kinase cascade. One effect of this pathwayis the increased production of the gene regulatory protein Myc. Mycincreases the transcription of several genes, including the geneencoding cyclin D and a subunit of the SCF ubiquitin ligase. Theresulting increase in G₁-Cdk and G₁/S-Cdk activities promotes Rbphosphyorylation and activation of the gene regulatory protein E2F,resulting in S-phase entry, in which G₁-Cdk activity initiates Rbphosphorylation, in turn inactivating Rb and freeing E2F to activate thetranscription of S-phase genes including the genes for a G₁/S-cyclin(cyclin E) and S-cyclin (cyclin A). The resulting appearance of G₁/S-Cdkand S-Cdk further enhances Rb phosphorylation, forming a positivefeedback loop, and the E2F acts back to stimulate the transcription ofits own gene, forming another positive feedback loop. Myc may alsopromote E2F activity directly by stimulating the transcription of theE2F gene. The result is the increased transcription of genes entry intoS phase. However if this pathway is overactive, it will cause cancercell growth.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the Ras-MAP kinase cascade so that the pathway is notoveractive.

The compound or its derivatives work in Ras-dependent or Myc pathway.Sometimes the mutation of amino acid in Ras causes the protein to becomepermanently overactive, stimulating the Ras-dependent signal pathwaysoveractive in absence of mitogenic stimulation. Similarly, mutationsthat cause an overexpression of Myc promote excessive cell growth, whichin turn promotes the development of cancer.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the components of the Ras-dependent or Myc pathway to make sureit is not overactive.

The compound or its derivatives reactivate the abnormal cell checkpointmechanism. Inside the cell, there is a checkpoint mechanism whichdetects abnormal mitogenic stimulation and causes abnormally overactivecells to go into apoptosis. However this mechanism is not active incancer cells due to mutations in the genes that encode essentialcomponents of the checkpoint responses. If the mutation happens in thecheckpoint mechanism, the cancer cell will grow and divide endlessly.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliareactivate the checkpoint mechanism to stop the cancer cell growth.

The compound or its derivatives affect the extracellular growthsignaling pathways. The extracellular growth factors that stimulate cellgrowth are bound to receptors on the cell surface and activateintracellular signaling pathways. It activates the enzyme PI3-kinase,which promotes protein synthesis, at least partly through the activationof EIF4e and phosphorylated S6 kinase, resulting in increased mRNAtranslation and then a stimulation of cell growth.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the components or receptor relate to extracellular growth. Itbinds the receptor of ovarian cancer cells so as to stop the cancer cellgrowth.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the components relating to Ras and MAP Kinase, which ceasesovarian cancer cell growth.

The compound or its derivatives affect the intracellular mechanism. Celldivision is also controlled by an intracellular mechanism that can limitcell proliferation. In normal cells, the Myc protein acts in the nucleusas a signal for cell proliferation. Large quantities of Myc can causethe cell to proliferate in excess and form a tumor.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the components or receptor of the Myc cell's proliferation tostop the tumor cells from dividing.

The compound or its derivatives affect the TGF-alpha signaling pathway.TGF-alpha is produced by keratincytes, macrophages, hepatocytes, andplatelets. Its synthesis is stimulated by the infection by viruses.TGF-Alpha induces the long term proliferation of murine and chickenimmature hematopoietic progenitor cell such as BFU-E without causingdifferentiation. It also induces the terminal differentiation ofBFU-Ecell into erythrocytes. TGF-Alpha stimulates the proliferation ofcultured endothelial cells. It plays an importance role in thevascularisation of tumor tissues.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the components or receptor of TGF-alpha to suppress ovariancancer and bladder cancer cell growth.

The compound or its derivative compounds affect the TGF-beta signalingpathway. TGF-beta regulates growth and proliferation of cells, blockinggrowth of many cell types. There are two TGF-beta receptors: Type 1 andType 2. They are serine-threonine kinases that signal through the SMAD(Protein named after the first two identified, Sma in C. elegans and Madin Drosophila) family of transcriptional regulators. The TGF-betapathway and mutation in SMADs are associated with cancer in humans.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the components or receptor of TGF-beta to suppress the ovariancancer and bladder cancer cell growth.

The compound or its derivatives reactivate the cell functions which aredamaged by DNA viruses. DNA tumor viruses cause cancer by interferingwith cell cycle control Rb protein and the p53 protein. Mutation in p53gene will allow cancer cells to survive and proliferate despite DNAdamage. The papillomanius uses the proteins E6 and E7 to release the p53and Rb respectively. This action activates mutated cells, allowing themto survive and then divide and accumulate. The accumulation of damagedcells can lead to cancer.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the proteins E6 and E7 and release the proteins Rb and p53,which will prevent abnormal cells from dividing. It also regulates orreacts with the protein, causing the cancer cells to die.

The compound or its derivatives affect the p53 signaling pathway. p53helps multi-cellular organisms cope safely with DNA damage and otherstressful cellular events, stopping cell proliferation in circumstanceswhere it would be dangerous. Cancer cells tend to contain largequantities of mutant p53 protein, suggesting that the genetic accidentsthey undergo or the stresses of growth in an inappropriate environmentcreated the signals that normally activate the p53 protein. Thus, theloss of p53 activity can be extremely dangerous in relation to cancerbecause it allows mutant cells to continue through the cell cycle. Italso allows them to escape apoptosis. So, if their DNA is damaged, somecells will die but the cells which survive will carry on dividingwithout pausing to repair the damage. This may cause the cells to die,or they could survive and proliferate with a corrupted genome, whichcould lead to loss of both tumor suppressor genes and the activation ofoncogenes, for example by gene amplification. Gene amplification couldenable cells to develop resistance against therapeutic drugs.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaregulate the components and receptor of the p53 pathway, which stops thecancer cells from dividing.

The compound or its derivatives affect the cell suicide signalingpathway. All cells with a nucleus contain various inactive procaspases,awaiting a signal before destroying the cell. Each suicide protease ismade as an inactive proenzyme called procaspase. It is usually activatedby proteolytic cleavage by another member of the caspase family. Two ofthe cleaved fragments come together to form the active part of thecaspase, and the active enzyme is thought to be a tetramer of two ofthese two parts. Each activated caspase molecule can cleave manyprocaspase molecules, which in turn activates more molecules. Through achain reaction or cascade, this leads to the explosive action of a largenumber of procaspase molecules. Then, some of the activated procaspasescleave a number of key proteins in the cell, including specificcytosolic proteins and nuclear-lamins leading to the controlled death ofthe cell.

Activating the death receptor on the outside of the cell can alsotrigger inactive procaspases. For example, killer lymphocytes can causeapoptosis by producing the protein Fas on the surface of the targetedcell. These clusters of Fas protein then recruit intracellular adaptorproteins that bind and aggregate procaspase-8 molecules. These thencleave and activate one another. The activated caspase-8 molecules thenactivate downstream procaspases to induce apoptosis.

However in cancer cells, the signal to destroy the cell is blocked, dueto gene mutation. This means that the cancer cells continue to divide,thereby causing a tumor.

Compounds or compositions derived from the plant Xanthoceras Sorbifoliaunblock the suicide signals, allowing cancer cells to destroythemselves.

Structure showed in FIG. 67.

This invention provides a method for inhibiting tumor cell growthcomprising contacting an amount of the above-described compound, whereinR1, R2, R3, R4 are short aliphatic chain and R5 contains an oxyl group;and a pharmaceutically acceptable carrier effective to inhibit growth ofsaid tumor cells.

This invention provides a method for inhibiting tumor cell growthcomprising contacting an amount of the above-described compounds.

This invention provides a method for inhibiting tumor cell growthcomprising contacting an amount of the compound comprising: a sugar; atriterpene or Sapogenin; side chain at Carbon 21 and 22 or Angeloylgroups, operatively linked form the compound; and a pharmaceuticallyacceptable carrier.

Structure showed in FIG. 67.

This invention provides a method for inhibiting tumor cell growth in asubject comprising administering to the above-described subject, whereinR1, R2, R3, R4 are short aliphatic chain and R5 contains an oxyl group;effective to inhibit growth of said tumor cells and a pharmaceuticallyacceptable carrier.

This invention provides a method of for inhibiting tumor cell whereinR1=R2=R3=R4=CH3 and R5 contains an oxyl bond.

This invention provides a method for inhibiting tumor cell growthcomprising contacting an amount of the above-described compounds.

This invention provides a compound consist of a triterpene orsapongenin, sugar moiety connected to the backbone. A sugar was linkedthe C21 position where two angeloyl groups were attached. This compoundhas the anti-cancer activity.

This invention provides a method for inhibiting tumor cell growthcomprising contacting an amount of the compound is a triterpene orsapongenin with any two of angeloyl group or tigloyl group or senecioylgroup or their combinations attach to carbon 21 and 22, or any two ofangloyl group or tigloyl group or senecioyl group or their combinationsattached to a sugar moiety which bonds to carbon 21 or 22.

The structures of this invention or its derivative can be synthesis orfrom biological sources.

This invention will be better understood from the examples which follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims which follow thereafter.

EXPERIMENTAL DETAILS

Experiment 1: Herb Extraction

(a) extracting Xanthoceras Sorbifolia powder of husks or branches orstems or leaves or kernels or roots or barks with organic solvent atratio of 1:2 for 4-5 times for 20-35 hours for each time to form anorganic extract; (b) collecting the organic extract; (c) refluxing theorganic extract for 2-3 times at 80° C. to form second extracts; (d)removing the organic solvent from the second extract; and (e) Drying andsterilizing the extract to form a Xanthoceras Sorbifolia extract powder.

Experiment 2: Analysis of Xanthoceras Sorbifolia Extract Components byHPLC Chromatography

Methods

HPLC. A C-18 reverse phase μbondapak column (Water P/N 27324) wasequilibrated with 10% acetonitrile, 0.005% Trifluoroacetic acid(equilibration solution). An extract of Xanthoceras Sorbifolia preparedusing the methods of the present invention was dissolved inequilibration solution (1 mg/ml) before being applied onto the column.20 ug of samples was applied into column. Elution conditions: Fractionswere eluted (flow rate 0.5 ml/min.) with acetonitrile (concentrationgradient from 10% to 80% in 70 min) and then remains at 80% for 10 min(70-80 min). The acetonitrile concentration then decreased to 10% (80-85min) and remained at 10% for 25 min (85-110 min). The fractions weremonitored at 207 nm and recorded in chart with a chart speed of 0.25cm/min and with a OD full scale of 0.128.

Instruments. Waters Model 510 Solvent Delivery System; Waters 484tunable Absorbance Detector; Waters 745/745B Data Module.

Absorbance analysis. The absorption profile of Xanthoceras Sorbifoliaextract at various wavelengths was determined. An extract of XanthocerasSorbifolia of the present invention was dissolved in 10%acetonitrile/TFA and scanned at 200-700 nm with a spectrophotometer[Spectronic Ins. Model Gene Sys2].

Results

HPLC. About 60-70 peaks can be accounted for in the profile. Among themfour are major peaks, 10 are medium size and the rest are smallfractions. The major peaks are labelled with a to z following increasedconcentration of acetonitrile elution. See FIG. 6.

Absorption maximum. Three absorption maximum were identified forXanthoceras Sorbifolia plant extract; 207 nm, 278 nm and 500 nm. SeeFIG. 41.

Experiment 3: Screening of Cytotoxicity of Xanthoceras SorbifoliaExtract with Cancer Cells Derived from Different Human Organs Using MTTAssay

Methods and Materials

Cells. Human cancer cell lines were obtained from American Type CultureCollection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460(lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2(liver), U2OS (bone), T98G (brain) and OVCAR-3 (ovary). Cells were grownin culture medium (HeLa-S3, DU1 45, MCF-7, Hep-G2 and T98G in MEN(Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI-1640; HCT-116, U2OSin McCoy-5A) supplemented with 10% fetal calf serum, glutamine andantibiotics in a 5% CO₂ humidified incubator at 37° C.

MTT assay. The procedure for MTT assay followed the method described in(Carmichael et al., 1987) with only minor modifications. Cells wereseeded into a 96-wells plate at concentrations of 10,000/well (HTB-9,HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2,U2OS), or 40,000/well (K562), for 24 hours before drug-treatment. Cellswere then exposed to drugs for 48 hours (72 hours for HepG2, U2OS, and96 hours for MCF-7). After the drug-treatment, MTT (0.5 mg/ml) was addedto cultures for an hour. The formation of formazan (product of thereduction of tetrazolium by viable cells) was dissolved with DMSO andthe O.D. at 490 nm was measured by an ELISA reader [Dynatech. ModelMR700]. The MTT level of cells before drug-treatment was also measured(T0). The % cell-growth (% G) is calculated as:% G=(TD−T0/TC−T0)×100  (1)

-   -   where TC or TD represent O.D. readings of control or        drug-treated cells. When T0>TD, then the cytotoxicity (LC)        expressed as % of the control is calculated as:        % LC=(TD−T0/T0)×100.

Results. Among the 10 cell lines studies, their sensitivity towardXanthoceras Sorbifolia extract can be divided into four groups (mostsensitive: Ovary. Sensitive: bladder, bone, prostate, and leukocyte,marginal sensitive: liver, breast, and brain; and lease sensitive:colon, cervix, and lung) (FIG. 8, 10A-D). Their IC50 values are listedin Table 3.1. TABLE 3.1 IC50 values of Xanthoceras Sorbifolia ExtractDetermined in Different Cancer Cells IC50 determined by Cancer cellsfrom different organs MTT assay (ug/ml) Ovary (most sensitive) 15-15Bladder (sensitive) 45-50 Bone 40-55 Prostate 40-50 Leukocyte 45-50Liver (marginal sensitive) 45-65 Breast  65 Brain 70-85 Colon (leastsensitive)  90 Cervix 115 Lung 110

Xanthoceras Sorbifolia plant extract stimulate cell growth of bladder,bone and lung cells. See FIGS. 10A, 10D.

To invest Among these cell line studied, it was found that lowconcentrations of the igate the growth and inhibition components of theXanthoceras Sorbifolia plant extract, the plant extract wasfractionated. FIG. 5 shows the results of the screening of cell growthactivity of fractions obtained after FPLC chromatography. The assay wasconducted with bladder cells. The fractions obtained from FPLC as shownin FIG. 20 were used. As shown in this figure, that different componentsof Xanthoceras Sorbifolia extracts cause either growth or inhibitioneffects on cells. Only fraction 5962 (Fraction Y) causes cellinhibition. Fractions 610 and 1116 cause minor stimulation of cellgrowth. Abscissa: concentration (ug/ml). Ordinate: % Cell Growth(determined by MTT assay).

Experiment 4: Purification of Inhibition Components in the XanthocerasSorbifolia Extract.

(A) Fractionation of Xanthoceras Sorbifolia Extracts Components withFPLC.

Methods

Column. Octadecyl functionalized silica gel; column dimension: 2 cm×28cm; equilibrated with 10% acetonitrile —0.005% TFA.

-   -   Sample loading: 1-2 ml, concentration: 100 mg/ml in 10%        acetonitrile/TFA.    -   Gradient elution: 10-80% acetonitrile in a total volume of 500        ml.    -   Monitor absorption wavelength: at 254 nm.    -   Fraction Collector: 5 ml/fractions (collect from 10% to 72%        acetonitrile, total 90 fractions)    -   Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results. The elution profile shows 4-5 broad fractions. See FIG. 20.These fractions were analyzed with HPLC. Specific components, i.e., a-zas specified in FIG. 6, are then assigned in the FPLC fractions.

FPLC fractions are grouped into 7 pools and analyzed for cell growthactivity with bladder cells with MTT assay. It was found only one pool(#5962) contains inhibition activity. See FIG. 5.

(B) Fractionation of Fraction #5962 with FPLC by a C18 Open Column with64% Acetonitrile Isocratic Elution

Methods

Column. Octadecyl-functionalized silica gel; 50 ml; 2 cm×28 cm;equilibrated with 64% acetonitrile —0.005% TFA.

-   -   Sample loading: 0.2 ml, with concentration: 1-2 mg/ml in 65%        acetonitrile/TFA.    -   Elution: 64% acetonitrile isocratic.    -   Monitor absorption wavelength: at 254 nm.    -   Fraction Collector: 1 ml fraction (collect the first 90        fractions)    -   Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results. Fraction 5962 was further separated with an open ODS-C18 columnusing isocratic 64% acetonitrile elution. Two major fractions, i.e., Xand Y, were collected. See FIG. 42. MTT assay showed that only the Yfraction has the inhibition activity. See FIG. 43.

(C) Analysis of Fraction Y with HPLC

Methods

Column. Waters μ-bondapak C18 (3.9 mm×300 cm).

-   -   Elution: 35% or 45% isocratic elution.    -   Flow rate: 0.5 ml/min; monitored at 207 nm with O.D. Scale of        0.128; chart speed: 0.25 cm/min.

Results. On 45% isocratic analysis, Three fractions of Y were obtained(FIG. 44).

(D) Final Isolation of Active Y Component with Preparative HPLC.

Methods

Column: A preparative HPLC column (Waters Delta Pak C18-300A);

-   -   Elution: 45% acetonitrile isocratic elution with flow rate of 1        ml/min.    -   Monitor at 207 nm;    -   Fractions (designated as Y1, Y2, etc.) were collected and        lyophilized.

Results. Final separation of Y fractions was achieved by HPLC with apreparative column (FIG. 7). These fractions (compound Y1, Y2, Y3 andY4) including the major fraction Y3 (designated as compound Y) werecollected. Re-chromatography of the Compound Y showed a single peak inHPLC with a C18 reverse phase column. See FIG. 7A.

(E) Appearance and solubility. The pure Compound Y is amorphous whitepowder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrileand 100% pyridine.

(F) Inhibition analysis of Compound Y with MTT Assay.

Inhibition analysis of Compound Y was determined with MTT assay. Theresults indicated that (a) Compound Y has activity against ovariancancer cells (OCAR-3) with IC50 value of 1.5 ug/ml which is 10-15 timesmore potent than the unpurified extract. See FIG. 3; and FIG. 8.

(b) Compound Y maintains its selectivity against ovarian cancer cellsversus cervical cancer cells (HeLa). See FIG. 9.

Experiment 5: Determination of the Chemical Structure of Compound Y ofXanthoceras Sorbifolia Extract.

Methods

NMR analysis. The pure compound Y of Xanthoceras Sorbifolia weredissolved in pyridine-D5 with 0.05% v/v TMS. All NMR spectra wereacquired using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe(¹H/¹³C/¹⁵N/³¹P) at 298 K. The numbers of scans for 1D ¹H spectra were16 to 128, depending on the sample concentration. 2D HMQC spectra wererecorded with spectral widths of 6000×24,000 Hz and data points of2024×256 for t₂ and t₁ dimensions, respectively. The numbers of scanswere 4 to 128. 2D HMBC were acquired with spectral widths of 6000×30,000Hz and data points of 2024×512 for t₂ and t₁ dimensions, respectively.The numbers of scans were 64. The 2D data were zero-filled in t1dimension to double the data points, multiplied by cosine-square-bellwindow functions in both t1 and t2 dimensions, and Fourier-transformedusing software XWIN-NMR. The final real matrix sizes of these 2D spectraare 2048×256 and 2048×512 data points (F2×F1) for HMQC and HMBC,respectively.

Mass spectral analysis. The mass of samples was analyzed by (A)MALDI-TOF Mass Spectrometry and by (B) ESI-MS Mass spectrometry. (A)Samples for MALDI-TOF were first dissolved in acetonitrile, then mixedwith the matrix CHCA (Alpha-cyano-4-hydroxycinnamic acid, 10 mg CHCA/mLin 50:50 water/acetonitrile and 0.1% TFA in final concentration). Themolecular weight was determined by the high resolution mass spectroscopeanalysis with standards. (B) For ESI, the sample was analyzed with LCQDECA XP Plus machine made by Thermo Finnigan. It is ionized with ESIsource and the solvent for the compound is acetonitrile.

Results. The profile of the proton NMR is presented in FIG. 11. The 2DNMR profiles of HMQC and HMBC are shown in FIGS. 12 and 13,respectively.

Table 5.1 summarizes the 2D NMR chemical shift data and shows theassignment of functional groups derived from these chemical shifts.Based on these data and analysis, the structure of compound Y isassigned and shown below.

Structure of Compound Y:

The chemical name of compound Y is:3-O-[β-D-galactopyranosyl(1→2)]α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene.TABLE 5.1 ¹³C and ¹H NMR Data for Compound Y (in Pyridine-d₅)^(a)Position C H Key HMBC correlations  1 38.7 0.83, 1.40 C-3, C-5, C-9  226.4 1.81, 2.14 —  3 89.6 3.25, 1H, dd, C-23, C-24, GlcA C-1′ 12.0/4.0Hz  4 39.4 — —  5 55.3 0.78 —  6 18.5 1.55, 1.59 C-8, C-10  7 36.5 2.00,2.10 C-5, C-9  8 41.2 — —  9 47.0 3.06 C-7, C-8, C-12, C-14, C- 26 1037.2 — — 11 23.7 1.74, 1.89 — 12 125.2 5.49, 1H, br s C-9, C-11, C-14,C-18 13 143.4 — — 14 47.5 — — 15 67.3 4.21 C-8, C-27 16 73.6 4.45 C-14,C-15, C-18 17 48.3 — — 18 40.8 3.07 C-12, C-13, C-14, C-16, C-19, C-20,C-28, 19 46.8 1.41, 1.69 — 20 36.2 — — 21 79.3 6.71, 1H, d, 10 Hz C-20,C-22, C-29, C-30, 21-O-Ang C-1′′′′ 22 73.5 6.32, 1H, d, 10 Hz C-16,C-17, C-21, C-28, 22-O-Ang C-1′′′′ 23 27.7 1.26, 3H, s C-3, C-4, C-5,C-24 24 16.5 1.16, 3H, s C-3, C-4, C-5, C-23 25 16.0 0.81, 3H, s C-1,C-5, C-9, C-10 26 17.3 0.99, 3H, s C-7, C-8, C-9, C-14 27 21.0 1.85, 3H,s C-8, C-13, C-14, C-15 28 62.9 3.50, 1H, d, 11.0 Hz, C-16, C-17, C-18,C-22 3.76, 1H, d, 11.0 Hz, 29 29.2 1.09, 3H, s C-19, C-20, C-21, C-30 3020.0 1.32, 3H, s C-19, C-20, C-21, C-29 GlcA  1′ 104.9 4.89, 1H, d, 7.8Hz C-3  2′ 79.1 4.38 GlcA C-1′, C-3′, Gal C-1″  3′ 86.1 4.20 GlcA C-2′,C-4′, Ara C-1′′′  4′ 71.5 4.42 GlcA C-3′, C-5′, C-6′  5′ 78.0 4.52 GlcAC-4′, C-6′  6′ 171.9 — — Gal  1″ 104.6 5.32, 1H, d, 7.7 Hz GlcA C-2′  2″73.6 4.42 Gal C-1″, C-3″  3″ 74.9 4.10 Gal C-2″  4″ 69.5 4.56 Gal C-2″,C-3″  5″ 76.4 3.94 Gal C-4″, C-6″  6″ 61.6 4.43, 4.52 Gal C-4″, C-5″Ara-f  1′′′ 110.6 6.03. 1H, br s GlcA C-3′, Ara C-2′′′, C-4′′′  2′′′83.4 4.94 Ara C-3′′′  3′′′ 78.3 4.78 Ara C-2′′′  4′′′ 85.2 4.82 AraC-5′′′  5′′′ 62.2 4.12, 4.28 Ara C-3′′′ 21-O-Ang  1′′′′ 167.7 — —  2′′′′129.6 — —  3′′′′ 137.2 5.96, 1H, dq, 7.0/1.5 Hz Ang C-1′′′′, C-4′′′′,C-5′′′′  4′′′′ 15.5 2.10, 3H, dq, 7.0/1.5 Hz Ang C-2′′′′, C-3′′′′  5′′′′20.8 2.00, 3H, s Ang C-1′′′′, C-2′′′′, C-3′′′′ 22-O-Ang  1′′′′ 167.9 — — 2′′′′ 129.8 — —  3′′′′ 136.3 5.78, 1H, dq, 7.0/1.5 Hz Ang C-1′′′′,C-4′′′′, C-5′′′′  4′′′′ 15.5 1.93, 3H, dq, 7.0/1.5 Hz Ang C-2′′′′,C-3′′′′  5′′′′ 20.5 1.74, 3H, s Ang C-1′′′′, C-2′′′′, C-3′′′′^(a)The data were assigned based on HMQC and HMBC correlations.

FIG. 14 and 15 show the mass spectrum of Compound Y as determined byMALDI-TOF and ESI-MS techniques. Based on these data, the mass ofcompound Y is 1140.57 which agrees with the theoretical mass of thecompound Y.

Conclusion

The active compound Y isolated from extract of Xanthoceras Sorbifolia isa triterpenoid saponins with three sugars and biangeloyl groups attachedto the C21 and C22 positions of the backbone. The formula of Y isC₅₇H₈₈O₂₃, and the chemical name of Compound Y is:3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene.

Experiment 6: Determination of the Chemical Structure of Compound Y1 ofXanthoceras Sorbifolia Extract.

Methods

The method for NMR and MS analysis for compound Y1 are same as describedin Experiment 5.

Results. The profile of the H—NMR of Y1 is presented in FIG. 16. The 2DNMR profiles of HMQC, HMBC and COSY are shown in FIGS. 17, 18 and 19,respectively.

Table 6.1 summarizes the 1D and 2D NMR chemical shift data and shows theassignment of functional groups derived from these chemical shifts.TABLE 6.1 ¹³C and ¹H NMR Data for Compound Y1 (in Pyridine-d₅)^(a)Position C H  1 38.6 0.85, 1.33  2 26.3 1.86, 2.10  3 89.7 3.25, 1H, dd 4 39.5 —  5 55.5 0.75  6 18.3 1.40, 1.43  7 33.1 1.20, 1.50  8 40.0 — 9 46.7 1.69 10 36.5 — 11 22.5 2.30 12 123.6 5.36, 1H, br s 13 143.5 —14 41.8 — 15 34.7 1.53, 1.73 16 68.5 4.45 17 48.2 — 18 39.9 3.04 19 47.61.30, 3.05 20 36.7 — 21 85.3 5.05, 1H, d 22 73.8 6.17, 1H, d 23 27.71.29, 3H, s 24 16.5 1.16, 3H, s 25 15.5 0.81, 3H, s 26 17.1 0.82, 3H, s27 20.6 1.83, 3H, s 28 63.7 3.42, 1H, d, 3.60, 1H, d 29 29.9 1.42, 3H, s30 19.9 1.37, 3H, s GlcA  1 105.0 4.88, 1H, d  2 79.0 4.37  3 86.0 4.20 4 71.6 4.43  5 78.0 4.50  6 171.8 — Gal  1 104.5 5.31, 1H, d  2 73.54.43  3 74.9 4.10  4 69.5 4.57  5 76.3 3.95  6 61.1 4.44, 4.53 Ara-f  1110.9 6.04, 1H, br s  2 83.3 4.95  3 78.3 4.78  4 85.2 4.82  5 62.04.13, 4.31 21-O-Rha  1 105.1 4.92, 1H, d  2 70.5 4.25  3 74.0 5.59  471.5 5.70  5 68.5 3.89  6 17.6 1.18, 3H, d Rh-3-Ang  1 167.2 —  2 127.9—  3 138.7 5.92, 1H, q  4 15.7 2.02, 3H, d  5 20.6 1.92, 3H, s Rh-4-Ang 1 167.2 —  2 128.0 —  3 137.9 5.87, 1H, q  4 15.5 1.96, 3H, d  5 19.81.85, 3H, s 22-O-Ac  1 171.4 —  2 21.8 2.31, 3H, s

.

Based on these data and analysis, the structure of compound Y1 isassigned and shown below.

The chemical name of Y1 is:

-   3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene.

Conclusion. Based on the chemical shift analysis, the active compound Y1isolated from extract of Xanthoceras Sorbifolia is a triterpenoidsaponins with four sugars and biangeloyl groups attached to the sugarmoiety. The formula of Y1 is C₆₅H₁₀₀0₂₇,

Results of Y2 Analysis

The profile of the proton NMR of Y2 is presented in FIG. 51.

The profiles of 2D NMR (HMQC) of Y2 is presented in FIG. 52.

Results of Y4 Analysis

The profile of the proton NMR of Y4 is presented in FIG. 53.

The profiles of 2D NMR (HMQC) of Y4 is presented in FIG. 54.

Experiment 7: Acid and Alkaline and Enzyme Hydrolysis of Compound Y.

(A) Removal of sugars from compound Y. Acid Hydrolysis of compound Ygenerates a compound with the following structure, designated herein asY-c: (FIG. 37).

Methods: 5 mg of compound Y is dissolved in 3 ml of MeOH and thentreated with 3 ml of 3N HCl. Hydrolysis of saponins will be conductedunder reflux for 4 hr. After hydrolysis, the solution will beneutralized with 5% Na₂CO₃ and extracted with Ethyl acetate three timesto afford an aqueous layer and an organic layer, containing sugars andaglycon, respectively. Aglycon from the organic layer will be furtherpurified on Silica gel chromatography in (CHCl₃: MeOH, 1:9) or with C18ODS HPLC chromatography. About 2 mg of compound with the above structureY-c can be obtained.

Method reference: Essentials of Carbohydrate Chemistry. By John F.Robyt, (Springer, 1998).

(B) Partial removal of sugars from compound Y. The linkage ofoligosaccharide can be cleaved by partial acid hydrolysis and byspecific enzyme hydrolysis. For example, the 1→4 linkage ofarabinofuranosyl can be removed by α-amylase. Other enzymes such asβ-amylase, isoamylase, glucose oxidase, mannanse and pullulanase can beused to cleave individual saccharide in saponins.

Coumpound Structure showed in FIG. 70.

Compound structure showed in FIG. 69.

Compound Structure showed in FIG. 68.

Experiment 8 Purification of Component R from Xanthoceras SorbifoliaExtract.

(A) Fractionation of Xanthoceras Sorbifolia Extracts Components withFPLC.

Methods

-   -   Column: Octadecyl functionalized silica gel; column dimension: 2        cm×28 cm; equilibrated with 10% acetonitrile—0.005% TFA.    -   Sample loading: volume: 1-2 ml, concentration: 100 mg/ml in 10%        acetonitrile/TFA.    -   Gradient elution: 10-80% acetonitrile in a total volume of 500        ml.    -   Monitored at 254 nm.    -   Fraction Collector: 5 ml/fraction.    -   Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results

The elution profile shows 4-5 broad fractions (FIG. 20). These fractionswere analyzed with HPLC. By comparison with the profiles of the originalsample, specific component, in this case the R component, is identifiedand then collected for further purification.

(B) Fractionation of R with FPLC with 30% Acetonitrile Isocratic Elution

Methods

-   -   Column: Octadecyl-functionalized silica gel; column dimemsion: 2        cm×28 cm; equilibrated with 30% acetonitrile —0.005% TFA.    -   Sample loading: 0.2 ml, with concentration: 1-2 mg/ml.    -   Elution: 30% acetonitrile isocratic. Monitor absorption        wavelength: at 254 nm.    -   Fraction Collector: 5 ml/fraction.    -   Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results

Fraction No. 39-41 from gradient elution of FPLC were pooled and furtherpurified with an open ODS-C18 column with isocratic 30% acetonitrileelution. Six identifiable fractions in two groups were collected. SeeFIG. 45. Fractions 6-13 were further characterized with HPLC.

(C) Analysis and Isolation of R with HPLC

Methods

-   -   Column: Waters μ-bondapak C18 (3.9×300 nm) and Waters DeltaPak        C18 (7.8 mm×30 cm).    -   Elution: Gradient (10-80%) and 30% isocratic elution.    -   Flow rate: 0.5 ml/min; monitored at 207 nm; with attenuation        0.128; chart speed: 0.25 cm/min.

Results

On HPLC gradient elution analysis, Fractions #9-11 contain a majorcomponent with a few minor components. See FIG. 46. These componentswere further separated into 4-5 components with the 30% acetonitrileisocratic elution in a DeltaPak column. The fraction designated hereinas “R1”, is the major component. See FIG. 47A. The pure R1 wassubsequently collected from the column elution. See FIG. 47B.

(D) Appearance and Solubility.

The pure R1 is amorphous white powder, soluble in aqueous alcohol(methanol, ethanol), 50% acetonitrile and 100% pyridine.

(E) Determination of the Chemical Structure of R1 Isolated fromXanthoceras Sorbifolia Extract

Methods

The NMR and MS Analysis of R1 are same as those described in Experiment5.

Results

The proton NMR profile of pure R1 is presented in FIG. 21. The 2D NMR(HMQC) spectra of R1 are presented in FIG. 22. The 2D NMR (HMBC) spectraof R1 are presented in FIG. 23. The 2D COSY spectrum is presented inFIG. 24. The Carbon 13 NMR spectrum is presented in FIG. 25.

Based on all the data presented above, Table 8.1 summarizes the resultsof the structural analysis and the assignment of the functional groupsof compound R1. TABLE 8.1 ¹³C and ¹H NMR Data for R1 (inPyridine-d₅)^(a) Position C H Key HMBC correlations  1 38.6 1.01, 1.63C-3, C-25  2 26.4 1.89, 2.33 C-3  3 89.1 3.26, 1H, dd, 12.0/4.2 Hz C-23,C-24, Glc′ C-1  4 39.2 — —  5 55.5 0.69, 1H, d, 11.4 Hz C-4, C-6, C-7,C-9, C-10, C-23, C-24, C-25  6 18.3 1.30, 1.39 C-5, C-8, C-10  7 32.51.41 C-6, C-26  8 40.1 — —  9 47.7 1.61 C-1, C-5, C-8, C-10, C-11, C-14,C-25, C-26 10 36.7 — — 11 23.7 1.90, 2.00 C-8, C-12, C-14 12 123.5 5.35,1H, br s C-9, C-14, C-18 13 142.9 — — 14 41.9 — — 15 25.7 1.88, 1.90 —16 18.0 1.95, 2.29 C-14, C-17, C-18 17 43.1 — — 18 41.6 2.60, dd,12.0/2.4 Hz C-19 19 46.3 1.28, 2.11 C-18, C-20, C-29, C-30 20 36.1 — —21 76.5 3.73, 1H, d, 9.6 Hz C-20, C-22, C-29, C-30 22 75.1 4.31, 1H, d,9.6 Hz C-16, C-17, C-21 23 27.9 1.20, 3H, s C-3, C-4, C-5, C-24 24 16.60.95, 3H, s C-3, C-4, C-5, C-23 25 15.7 0.95, 3H, s C-1, C-5, C-9, C-1026 16.7 1.07, 3H, s C-7, C-8, C-9, C-14 27 26.1 1.25, 3H, s C-8, C-13,C-14 28 75.8 4.10, 2H, br s C-16, C-17, C-18, C-22, Glc′′′ C-1 29 30.21.22, 3H, s C-19, C-20, C-21, C-30 30 19.5 1.26, 3H, s C-19, C-20, C-21,C-29 3-Glc′  1 106.5 4.84, 1H, d, 7.2 Hz C-3, Glc′ C-5  2 73.2 3.99 —  379.1 5.90, 1H, t, 9.6 Hz Ang C-1, Glc′ C-2, C-4  4 69.4 4.26 Glc′ C-6  576.4 3.95 Glc′ C-1, C-3  6 69.8 4.40, 4.83 Glc′ C-4, Glc″ C-1 Ang  1167.9 — —  2 128.7 — —  3 136.7 5.80, 1H, ddd, 6.6/0.6 Hz Ang C-1, C-4,C-5  4 15.7 1.93, 3H, dd, 6.6/0.6 Hz Ang C-2, C-3  5 20.6 1.81, 3H, sAng C-1, C-2, C-3 Glc″  1 105.4 5.09, 1H, d, 7.8 Hz Glc′ C-6, Glc″ C-5 2 74.9 4.05 Glc″ C-1  3 78.2^(b) 4.22 Glc″ C-5  4 71.4^(c) 4.24 Glc″C-6  5 78.3^(d) 3.90 —  6 62.4^(e) 4.40, 4.51 Glc″ C-4 28-Glc′′′  1103.5 4.72, 1H, d, 7.2 Hz C-28  2 75.3 4.22 Glc′′′ C-1, C-3, Rha C-1  379.8 4.25 —  4 71.6 4.20 —  5 76.6 4.13 Glc′′′ C-3  6 70.0 4.67, 2H, d,10 Hz Glc′′′′ C-1, Glc′′′ C-4, C-5 Rha  1 100.7 6.52, 1H, br s Glc′′′C-2, Rha C-3, C-5  2 72.3^(f) 4.70, d, 3.0 Hz Rha C-4  3 72.3^(f) 4.63,dd, Rha C-2  4 74.1 4.35 Rha C-2, C-5  5 69.1 4.79 —  6 18.7 1.82, 3H,d, 6.6 Hz Rha C-4, C-5 Glc′′′′  1 105.5 5.01, 1H, d, 7.8 Hz Glc′′′ C-6,Glc′′′′ C-5  2 74.9 4.05 Glc′′′′ C-1  3 78.2^(b) 4.22 Glc′′′′ C-5  471.4^(c) 4.24 Glc′′′′ C-6  5 78.3^(d) 3.90 —  6 62.5^(e) 4.40, 4.51Glc′′′′ C-4^(a)The data were assigned based on COSY, HMQC and HMBC correlations.^(b,c,d,f)The data with the same labels in each column were overlapped.^(e)The data with the same labels in each column may be interchanged.

Conclusion

Based on the chemical shift analysis, the compound R1 isolated fromextract of Xanthoceras Sorbifolia is a triterpenoid saponins with fivesugars and one angeloyl group attached to the sugar moiety. The chemicalstructure of R1 is:

The formula of compound R1 is C₆₅H₁₀₆O₂₉, and the chemical name of R1is:3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene

Experiment 9: Purification of component-O from Xanthoceras SorbifoliaExtract

(A) Fractionation of Xanthoceras Sorbifolia Extracts Components withFPLC

Methods:

-   -   Column: Octadecyl functionalized silica gel; column dimension: 2        cm×28 cm; equilibrated with 10% acetonitrile—0.005% TFA.    -   Sample loading: 1-2 ml, concentration: 100 mg/ml in 10%        acetonitrile/TFA.    -   Gradient elution: 10-80% acetonitrile in a total volume of 500        ml.    -   Monitor absorption wavelength: at 254 nm.    -   Fraction Collector: 5 ml/fraction.    -   Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.

Results.

The elution profile shows 4-5 broad fractions (FIG. 20). These fractionswere analyzed with HPLC. By comparison with the profiles of the originalsample, specific component, in this case the component-O, is identified(#28-30) and were collected for further purification.

(B) Purification of Component-O with HPLC with 20% AcetonitrileIsocratic Elution.

Methods.

-   -   Column: A preparative HPLC column (Waters Delta Pak C18-300A);    -   Samples: Fraction #28-30 from the gradient elution of FPLC were        pooled and applied into the HPLC.    -   Elution: 20% acetonitrile isocratic with flow rate of 1 ml/min.        Fractions were collected.    -   Monitored at 207 nm;    -   Fractions of interested were collected and lyophilized.

Results.

Sixteen identifiable fractions were observed in the elution profiles(FIG. 48). Fractions 28, 34 and 54 were further characterized with HPLCusing same condition (FIGS. 49 and 50). As show in these figures, asingle peak elution of fractions 28, 34 and 54 was obtained, indicatingthat they are homogeneous fractions (pure). These purified componentsare named as Compound O28, O34 and O54, respectively.

Appearance and solubility: The purified O-23 and O-34 are light yellowamorphous powder, soluble in aqueous alcohol (methanol, ethanol), 50%acetonitrile and 100% pyridine. The purified O-54 is white amorphouspowder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrileand 100% pyridine.

(C) Structure Analysis of Compound-O54.

Methods: The NMR and MS analysis of O54 are same as those described inExperiment 5.

Results

The profile of the proton NMR of compound-O54 is presented in FIG. 28.The 2D NMR (HMQC) spectra of 054 is presented in FIG. 29. The 2D NMR(HMBC) spectra of O54 presented in FIG. 30.

Based on all the data presented above, Table 9.1 summarizes the resultsof the structural analysis and the assignment of the functional groupsof compound-O54. TABLE 9.1 ¹³C and ¹H NMR data for 054 (inPyridine-d₅)^(a) Position C H Key HMBC correlations  1 38.6 1.03, 1.60C-3, C-25  2 26.3 1.90, 2.40 C-3  3 89.1 3.30, 1H, dd, 12.0/4.8 Hz C-23,C-24, Glc′ C-1  4 39.3 — —  5 55.6 0.70, 1H, d, 12.0 Hz C-4, C-6, C-7,C-9, C-10, C-23, C-24, C-25  6 18.5 1.30, 1.40 C-5, C-8, C-10  7 32.51.40 C-6, C-26  8 40.2 — —  9 47.8 1.59 C-1, C-5, C-8, C-10, C- 11,C-14, C-25, C-26 10 36.6 — — 11 23.6 1.90, 1.95 C-8, C-12, C-14 12 123.55.34, 1H, br s C-9, C-14, C-18 13 142.8 — — 14 41.9 — — 15 25.8 1.90 —16 18.1 1.90, 2.31 C-14, C-17, C-18 17 43.2 — — 18 41.7 2.58, dd,13.2/2.0 Hz C-19 19 46.5 1.28, 2.08 (t, 1H, t, C-18, C-20, C-29, C-3013.2 Hz) 20 36.2 — — 21 76.6 3.73, 1H, d, 10.2 Hz C-20, C-22, C-29, C-3022 75.1 4.31, 1H, d, 10.2 Hz C-16, C-17, C-21 23 27.9 1.22, 3H, s C-3,C-4, C-5, C-24 24 16.8 0.98, 3H, s C-3, C-4, C-5, C-23 25 15.8 0.95, 3H,s C-1, C-5, C-9, C-10 26 16.9 1.07, 3H, s C-7, C-8, C-9, C-14 27 26.11.26, 3H, s C-8, C-13, C-14 28 75.9 4.10, 2H, br s C-16, C-17, C-18,C-22, Glc′′′ C-1 29 30.1 1.24, 3H, s C-19, C-20, C-21, C-30 30 19.61.26, 3H, s C-19, C-20, C-21, C-29 3-Glc′  1 106.5 4.86, 1H, d, 7.8 HzC-3, Glc′ C-5  2 74.9 3.99 —  3 78.2^(b) 4.20 —  4 70.8 4.36 —  5 76.53.95 —  6 70.5 4.40, 4.88 (d, 1H, 9.6 Hz) Glc″ C-1 Glc″  1 105.5 5.12,1H, d, 7.8 Hz Glc′ C-6, Glc″ C-5  2 75.0^(c) 4.03 Glc″ C-1  3 78.2^(b)4.20 —  4 71.5^(d) 4.20 —  5 78.3^(e) 3.91 —  6 62.4^(f) 4.40, 4.48 Glc″C-4, C-5 28-Glc′′′  1 103.6 4.72, 1H, d, 7.2 Hz C-28  2 75.3 4.22 Glc′′′C-1, C-3, Rha C-1  3 79.8 4.21 —  4 71.6 4.19 —  5 76.8 4.14 G1c′′′ C-3 6 70.3 4.67, 2H, d, 10.2 Hz Glc′′′′ C-1, Glc′′′ C-4, C-5 Rha  1 100.66.51, 1H, br s Glc′′′ C-2, Rha C-3, C-5  2 72.3^(g) 4.72, d, 3.0 Hz RhaC-4  3 72.3^(g) 4.61, dd, 10.8/3.6 Hz, Rha C-2  4 74.1 4.36 Rha C-2, C-5 5 69.2 4.77 —  6 18.6 1.80, 3H, d, 6.0 Hz Rha C-4, C-5 Glc′′′′  1 105.65.00, 1H, d, 7.8 Hz Glc′′′ C-6, Glc′′′′ C-5  2 75.0^(c) 4.03 Glc′′′′ C-1 3 78.2^(b) 4.20 —  4 71.5^(d) 4.20 —  5 78.3^(e) 3.91 —  6 62.5^(f)4.40, 4.48 Glc′′′′ C-4, C-5^(a)The data were assigned based on COSY, HMQC and HMBC correlations.^(b,c,d,e,g)The data with the same labels in each column wereoverlapped.^(f)The data with the same labels in each column may be interchanged.

Conclusion

Based on the chemical shift analysis, the compound O54 isolated fromextract of Xanthoceras Sorbifolia is a triterpenoid saponins with fivesugars. The chemical structure of Compound O54 is:

The structure of compound 054 is presented in the following figure.

The formula of compound O54 is C₆₀H₁₀₀O₂₈, and the chemical name of O54is: The chemical name of compound-O54 is:3-O-β-D-glucopyranosyl-(1→6)]-βD-glucopyranosyl-28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,21β,22α,28-tetrahydroxyolean-12-ene.

Results of O28 Analysis

The profile of the proton NMR of O28 is presented in FIG. 55.

The profiles of 2D NMR (HMQC) of O28 is presented in FIG. 56.

Results of O34 Analysis

The profile of the proton NMR of O34 is presented in FIG. 57.

The profiles of 2D NMR (HMQC) of O34 is presented in FIG. 58.

Experiment 11: Extracts X and Y from the Wenguanguo Plant (XanthocerasSorbifolia) Improve Learning in Normal Aging Mice

Extracts X and Y are Different Wenguanguo Extracts

Aging male mice at 16 months of age weighing 35-55 gm were trained in aSMG-2 filled with water 11 cm deep (25-26° C.). The SMG-2 had a startpoint, 4 blind terminals, the escape platform and their routes. The micewere trained to find the escape platform, and the escape latencies fromthe water and error frequencies were recorded. After training 3 times,the mice which escaped from the water in 2 minutes were selected for thetest. The selected aging mice were divided into 9 groups of 11:1)control: receiving normal saline (NS); 2) Positive control: receivingXi-en-kai 0.9 g/kg; 3) X-I group: receiving 100 mg/kg4; 4) X-II group:receiving 200 mg/kg; 5) X-III group: receiving 400 mg/kg; 6) Y-I group:receiving 125 mg/kg; 7) Y-II group: receiving 250 mg/kg; 8) Y-III group:receiving 500 mg/kg and 9) model group.

All the drugs were received through oral administration, 20 ml/kg, 3, 6and 9 days before the water maze test. The escape latencies (EL) fromthe water and error frequencies were recorded. All data were analyzedwith t-test.

11.1. After 3 days of administration of extracts X and Y from WenguanguoPlant, hereinafter as “X” and “Y”, respectively, the escape latency inthe water maze by the group 9 mice receiving 500 mg/kg of Y decreasedsignificantly compared with the control (P<0.05). The other dosagetreatments showed improvement too but not significant ones. See Table11.1. TABLE 11.1 The Learning Effects of Plant Extracts afterAdministration in Aging Mice for 3 days Blind Blind Blind Blind GroupTerminal 1 Terminal 2 Terminal 3 Terminal 4 Error Frequency EL(sec)control 2.12 ± 2.27 3.62 ± 1.63 1.37 ± 2.41 2.68 ± 2.52 9.81 ± 3.4264.12 ± 24.8  positive 1.09 ± 1.57  2.0 ± 0.89  2.0 ± 1.26  2.0 ± 2.647.09 ± 2.30 39.45 ± 16.32 X I 1.18 ± 1.4  3.36 ± 2.65 2.27 ± 2.14 0.81 ±1.07 7.63 ± 4.47 51.72 ± 17.23 X II  2.2 ± 1.61  2.5 ± 1.51  2.0 ± 1.69 2.8 ± 1.03  9.5 ± 4.03  50.3 ± 20.84 X III 1.45 ± 2.33 2.72 ± 2.64 2.09± 2.11 1.81 ± 1.94 8.09 ± 4.67 46.91 ± 19.18 Y I 1.36 ± 1.91  3.0 ± 1.943.45 ± 3.2  1.09 ± 1.44 8.18 ± 3.78 46.36 ± 22.33 Y II 1.63 ± 1.80 3.81± 1.94 2.36 ± 1.12  .36 ± 1.50 10.18 ± 3.02  48.36 ± 20.61 Y III 2.18 ±3.34 1.63 ± 1.21 1.54 ± 1.29 1.81 ± 1.40 7.18 ± 4.30  41.45 ± 16.48*P < 0.05*

11.2. After 6 days of administration of X and Y, the error frequency inthe water maze in the mice with all dosage treatments decreasedsignificantly (P<0.05, P<0.01). The escape latency in the water maze bythe group 9 mice receiving 500 mg/kg of Y decreased significantly(P<0.05). See Table 11.2. TABLE 11.2 The Learning Effects of PlantExtract after Administration in aging mice for 6 days Blind Blind BlindBlind Error Group Terminal Terminal Terminal Terminal Frequency EL(sec)Control 2.74 ± 2.98  4.0 ± 2.67 3.26 ± 3.02  3.0 ± 2.67 12.9 ± 4.2060.58 ± 24.6  positive 1.64 ± 2.06 3.73 ± 2.05 1.18 ± 1.47  2.0 ± 1.90 8.55 ± 4.61* 38.64 ± 13.68 X I  1.7 ± 1.49  3.1 ± 2.02  1.8 ± 2.10  1.9± 1.66  8.5 ± 1.65*  47.7 ± 11.13 X II 1.18 ± 1.66  3.5 ± 2.02  2.0 ±1.73 1.73 ± 2.05  8.45 ± 3.14*  49.0 ± 13.29 X III 1.09 ± 1.30 3.55 ±2.07 1.91 ± 1.58 1.55 ± 1.44  8.0 ± 2.32* 46.36 ± 13.31 Y I  1.0 ± 1.673.09 ± 1.58 2.64 ± 1.8  1.54 ± 2.02  8.36 ± 3.07* 57.27 ± 19.88 Y II1.36 ± 1.50 3.36 ± 2.06  2.0 ± 1.67 1.18 ± 1.17  7.91 ± 3.05** 47.55 ±22.93 Y III  1.2 ± 1.14  4.1 ± 1.79  2.5 ± 2.84  1.4 ± 1.90  9.0 ± 3.23* 39.9 ± 8.56*P < 0.05*P < 0.01**

11.3. After 9 days of administration of X and Y, the error frequency inthe water maze in the mice with all dosage treatments decreasedsignificantly (P<0.05, P<0.01). The escape latency in the water maze bythe group 9 mice receiving 500 mg/kg of Y decreased significantly(P<0.05). See Table 11.3. FIG. 60 a, 60 b. TABLE 11.3 The LearningEffects of Plant Extracts after Administration in aging mice for 9 daysBlind Blind Blind Blind Group Terminal 1 Terminal 2 Terminal 3 Terminal4 Error Frequency EL(sec) Control 2.36 ± 1.65 2.64 ± 1.69 2.86 ± 2.54 1.5 ± 2.18 9.71 ± 3.52 59.71 ± 29.42 positive 1.64 ± 1.80 2.18 ± 1.541.55 ± 1.37 1.64 ± 2.20  7.0 ± 2.19* 33.36 ± 10.87 X I  1.1 ± 1.29  2.5± 2.37  1.9 ± 0.74  1.0 ± 1.15  6.5 ± 3.27* 40.8 ± 20.4 X II 1.18 ± 1.172.18 ± 1.60 1.36 ± 1.36 2.18 ± 1.25  6.91 ± 3.27* 42.73 ± 15.82 X III 1.0 ± 1.25  1.9 ± 1.79  1.3 ± 1.25  2.2 ± 1.16  6.4 ± 2.84*  35.1 ±11.76* Y I 1.82 ± 1.33 1.64 ± 1.69 1.82 ± 1.33 1.82 ± 1.47  7.09 ± 2.47*42.09 ± 20.93 Y II  1.2 ± 1.32  1.9 ± 1.37  2.6 ± 1.58  1.2 ± 1.32  7.1± 1.52*  34.4 ± 13.47* Y III  0.8 ± 1.03  2.5 ± 1.43  1.8 ± 1.40  2.0 ±1.70  7.0 ± 1.41*  31.9 ± 9.87**P < 0.05*P < 0.01**

The results indicated that the extracts X and Y had positive effects onimproving acquisition and retention of the tested aging mice. Inaddition, the effects increased with the period of receiving theextracts of X and Y prolonged.

Experiment 12: Effects of Extracts X and Y on Improving ImpairmentInduced by Pentobarbital Sodium in Water Maze Learning

12.1 After 10 days of administration of the extracts X and Y, theadministrated mice were injected with pentobarbital sodium to induceamnesia.

After 1 day administration of pentobarbital sodium, the results of watermaze learning showed that the time spent searching the terminal platformin the water maze by the mice receiving 100 mg/kg of X, and 125 mg, 250mg/kg and 500 mg/kg of Y decreased significantly (P<0.05).

Error frequency made in the water maze by the mice receiving 500 mg/kgof Y decreased significantly (P<0.05). See Table 12.1. TABLE 12.1Results of Water Maze Learning (First Day after Injection withPentobarbital) Blind Blind Blind Blind Group Terminal 1 Terminal 2Terminal 3 Terminal 4 Error Frequency EL(sec) control 0.92 ± 0.76 1.46 ±0.97 1.85 ± 1.07 1.23 ± 0.73 5.38 ± 2.33  63.0 ± 25.31 positive 0.69 ±0.70  1.3 ± 0.95  1.1 ± 0.74  1.1 ± 0.74  4.1 ± 1.85   36.5 ± 15.76** XI  0.5 ± 0.53  1.7 ± 0.82  1.2 ± 0.92  0.9 ± 0.32  4.2 ± 1.62  42.2 ±18.83* X II  0.9 ± 0.88  1.4 ± 0.70  1.6 ± 1.35  1.1 ± 0.88  5.0 ± 2.49 53.8 ± 16.10 X III  0.9 ± 0.74  1.7 ± 0.82  1.8 ± 0.42  0.9 ± 0.57  5.4± 1.58  58.1 ± 16.11 Y I  1.0 ± 0.89 1.36 ± 0.81 1.27 ± 1.01 0.73 ± 0.654.09 ± 2.02  42.73 ± 16.17* Y II  0.9 ± 0.74  1.7 ± 0.82*  1.0 ± 0.82* 0.6 ± 0.70*  4.2 ± 1.87   38.4 ± 15.19** Y III  0.6 ± 0.70  0.8 ± 0.63 1.4 ± 1.35  0.8 ± 0.63  3.6 ± 1.26*   38.5 ± 13.81**P < 0.05*P < 0.01**

12.2. After two days of injected of pentobarbital sodium, the time spentsearching the terminal platform and the error frequency made in thewater maze by all groups of mice receiving X and Y decreasedsignificantly (P<0.05). See Table 12.2. TABLE 12.2 Results of Water MazeLearning (Second Day after Injection with Pentobarbital) Blind BlindBlind Blind Group Terminal 1 Terminal 2 Terminal 3 Terminal 4 ErrorFrequency EL(sec) control 1.36 ± 0.50 1.86 ± 0.53 1.29 ± 1.07 1.14 ±0.66 5.64 ± 1.91  59.5 ± 34.95 positive  0.8 ± 0.63  1.8 ± 0.42  1.1 ±0.74  0.6 ± 0.70  4.3 ± 0.95*  34.7 ± 11.45* X I  0.9 ± 0.57*   1.0 ±0.67**  1.2 ± 0.79  0.8 ± 0.63  3.9 ± 1.73*  34.5 ± 12.67* X II  0.8 ±0.79*  1.5 ± 0.71  1.1 ± 0.88  0.5 ± 0.53*  3.8 ± 1.40*  35.9 ± 12.70* XIII  1.0 ± 1.05  1.3 ± 0.48*  1.3 ± 0.82  0.5 ± 0.53*  4.1 ± 1.79*  36.1± 11.10* Y I 1.09 ± 0.94 1.45 ± 0.52 0.91 ± 0.83 0.73 ± 0.65  4.18 ±1.08*  36.64 ± 14.38* Y II  0.9 ± 0.57*  1.3 ± 0.82  1.1 ± 0.88  0.8 ±0.63  4.1 ± 1.45*  35.5 ± 14.27* Y III  0.8 ± 0.63*   0.9 ± 0.74**  0.9± 0.57  0.9 ± 0.57   3.4 ± 1.43**  32.1 ± 13.12*P < 0.05*P < 0.01**

12.3. After three days of administration of pentobarbital sodium, thetime spent searching the terminal platform in the water maze by allgroups of mice receiving X and Y decreased significantly (P<0.05). Theerror frequency made in the water maze by the mice receiving 250 mg/kgand 500 mg/kg of Y decreased significantly (P<0.05). See Table 12.3,FIG. 61 a and FIG. 61 b. TABLE 12.3 Results of Water Maze Learning(Third day after Injection with Pentobarbital) Blind Blind Blind BlindGroup Terminal 1 Terminal 2 Terminal 3 Terminal 4 Error FrequencyEL(sec) control 0.85 ± 0.99  1.0 ± 0.58 1.46 ± 1.05 0.62 ± 0.65 3.92 ±1.75 48.92 ± 23.49 positive  0.4 ± 0.52  0.7 ± 0.48  0.9 ± 0.74  0.6 ±0.70  2.6 ± 0.97*  28.4 ± 13.78 X I  0.6 ± 0.70  0.8 ± 0.63  0.6 ± 0.70* 0.8 ± 0.92  2.8 ± 1.69  28.0 ± 17.10* X II  0.4 ± 0.52  1.0 ± 0.47  1.0± 0.82  0.6 ± 0.84  3.0 ± 1.25  32.0 ± 12.36* X III  0.5 ± 0.71  0.8 ±0.63  1.0 ± 0.82  0.9 ± 0.74  3.2 ± 1.23  31.8 ± 12.48* Y I  0.5 ± 0.53 0.7 ± 0.48  1.0 ± 0.67  0.6 ± 0.52  2.8 ± 0.63  31.0 ± 9.66* Y II  0.5± 0.53  0.6 ± 0.52  0.8 ± 0.42  0.6 ± 0.52  2.5 ± 0.53*  29.0 ± 7.80* YIII  0.3 ± 0.48  1.0 ± 0.47  0.9 ± 0.57  0.4 ± 0.52  2.6 ± 0.84*  30.2 ±7.54*P < 0.05*

The results indicated that the extracts X and Y had distinct positiveeffects on improving spatial learning and retention impairment inducedby pentobarbital sodium.

Experiment 13: Effects of Extracts X and Y on Improving ImpairmentInduced by Scopolamine Hydrobromide in Passive Avoidance

ICR mice weighing 16-20 gm were trained in a STT-2. A mouse was placedon the platform and the SDL were recorded. When the mouse stepped downand all four feet were on the grid, it received electric shock (36 V)immediately, and the EL was recorded. The mice with SDL and EL within2-60 seconds were selected for the test. The selected aging mice weredivided into 9 groups. Each group had 5 male and 5 female mice. All thedrugs were received with oral administration, 20 ml/kg, 3, 6 and 9 daysbefore the platform training. The SDL, E1 and error frequency (the timesof receiving electric shocks) made in 5 minutes were recorded. After 10days of administration all the groups of mice received Scopolaminehydrobromide by injection, 3 mg/kg. After 30 minutes of administrationof Scopolamine hydrobromide the mice were trained on the platform andthe training was repeated the next day. The performances of the mice inthe training were recorded. The SDL, EL and error frequency wererecorded. All data were analyzed with t-test.

13.1. After 9 days of administration of X and Y, the results of passiveavoidance in aging mice showed that the EL and the error frequency madeby the mice received the X and Y in all doses reduced. See Table 13.1.TABLE 13.1 Passive Avoidance in Aging Mice after 9 days ofAdministration Group EL (sec) SDL (min) Error Frequency (in 5 min)control 6.8 ± 5.93 131.5 ± 106.50 2.5 ± 1.47 positive 5.3 ± 3.74 148.6 ±81.26 1.7 ± 1.25 X I 7.0 ± 5.46 143.3 ± 19.77 1.8 ± 0.92 X II 6.2 ± 5.71141.1 ± 109.89 2.0 ± 1.25 X III 4.9 ± 3.70 145.7 ± 107.0 1.9 ± 1.52 Y I5.1 ± 3.95 153.0 ± 123.52 1.6 ± 1.33 Y II 3.9 ± 2.77 162.7 ± 108.92 1.8± 1.48 Y III 5.7 ± 3.12 159.4 ± 83.20 1.9 ± 0.93

13.2. After 10 days of administration of the X and Y, the tested micewere administrated with scopolamine. The results of passive avoidance inaging mice showed that the error frequency made in the passive avoidanceby the mice receiving 400 mg/kg of X, 250 mg/kg and 500 mg/kg of Yreduced significantly (P<0.05, P<0.01). See Table 13.2. TABLE 13.2Effects of plant extract X and Y on improving impairment induced byScopolamine Group EL (sec) Error Frequency (5 min) control 3.4 ± 3.030.9 ± 1.29 model 4.4 ± 4.09 1.6 ± 1.35 positive 3.1 ± 1.20 0.4 ± 0.52* XI 3.1 ± 2.08 0.8 ± 1.03 X II 3.7 ± 3.06 0.9 ± 1.52 X III 2.8 ± 1.48 0.3± 0.48** Y I 3.2 ± 2.49 0.7 ± 1.06 Y II 2.5 ± 0.97 0.5 ± 0.71* Y III 2.5± 0.71 0.5 ± 0.71**p < 0.05**p < 0.01

The results of passive avoidance test impaired mice by scopolamineshowed that the error frequency made by the mice receiving X and Y inall doses reduced significantly (P<0.05). The SDL prolongedsignificantly in mice receiving 250 mg/kg of Y.

The results indicated that the extracts X and Y had distinct positiveeffects on improving learning and retention impairment induced byscopolamine. See Table 3.3. TABLE 13.3 Effects of plant extract X and Yon improving impairment induced by Scopolamine. Group SDL (sec) ErrorFrequency (5 min) control 230.4 ± 96.61 0.7 ± 1.06 model 216.2 ± 100.771.5 ± 1.35 positive 286.0 ± 34.38* 0.4 ± 0.70* X I 245.7 ± 114.48 0.4 ±0.84* X II 260.4 ± 87.14 0.4 ± 0.84* X III 266.8 ± 65.64 0.5 ± 0.71* Y I252.7 ± 101.11 0.4 ± 0.84* Y II 285.8 ± 29.21* 0.4 ± 0.70* Y III 277.4 ±47.62 0.4 ± 0.70**p < 0.05

The results indicated that the extracts X and Y had distinct positiveeffects on improving learning and retention impairment induced byscopolamine hydrobromide.

Experiment 14: Effects of Extracts X and Y on Improving ImpairmentInduced by NaNO₂ in Water Maze Learning

ICR male mice weighing 16-19 gm were trained in a SMG-2 filled withwater 11 cm deep (25-26° C.). The SMG-2 has a start point, 4 blindterminals, the escape platform and their routes. The mice were trainedto find the escape platform, and the escape latencies from the water anderror frequencies were recorded. After training, the mice which escapedfrom the water within 1 minute were selected for the test. The selectedaging mice were divided into 9 groups of 11 mice. All the drugs werereceived with oral administration, 20 ml/kg, 3, 6 and 9 days before thewater maze test. After 10 days of administration all the groups of micewere received with NaNO₂ by injection, 120 mg/kg. After 24 hours ofadministration of NaNO₂ the mice were trained to find the escapeplatform, and the escape latencies from the water and error frequenciesmade in 2 minutes were recorded. The administration of pentobarbitalsodium continued for 3 days and the performances of the mice in watermaze test were recorded. The escape latencies (EL) from the water anderrors frequencies were recorded. All data were analyzed with t-test.

14.1. After 3 days of administration of X and Y the escape latency fromthe water maze and error frequency by the mice receiving X and Yreduced, although not significantly. See Table 14.1. TABLE 14.1 Effectsof Extracts X and Y on Water Maze Learning in Mice after 3 Days ofAdministration Blind Blind Blind Blind Group Terminal 1 Terminal 2Terminal 3 Terminal 4 Error Frequency EL(sec) control 1.05 ± 0.49 1.82 ±0.66 1.09 ± 0.75 0.86 ± 0.47 4.82 ± 0.96 48.27 ± 21.47 positive 0.91 ±0.30 1.27 ± 0.65 0.82 ± 0.60 0.64 ± 0.50  3.64 ± 0.92** 36.27 ± 11.83 XI 1.36 ± 0.81 1.73 ± 0.79 1.09 ± 0.83 0.55 ± 0.52 4.73 ± 2.05 37.82 ±14.24 X II 0.91 ± 0.30 1.82 ± 0.40 1.09 ± 0.94 1.00 ± 0.89 4.91 ± 1.5136.46 ± 11.97 X III 1.09 ± 0.54 1.45 ± 0.52 0.91 ± 0.70 0.45 ± 0.52 3.91± 0.70 36.46 ± 11.78 Y I 1.55 ± 0.52 1.82 ± 0.40  1.0 ± 0.89 0.45 ± 0.694.82 ± 1.33 41.46 ± 16.37 Y II 0.91 ± 0.30 1.18 ± 0.60 1.27 ± 1.10 0.73± 0.79 4.09 ± 2.21 36.82 ± 20.61 Y III 0.91 ± 0.30 1.55 ± 0.82 0.45 ±0.52 0.82 ± 0.40 3.73 ± 1.27 37.55 ± 13.85*P < 0.05**P < 0.01

14.2. After 6 days of administration of X and Y the escape latency fromthe water maze by the mice receiving 400 mg/kg of X and 500 mg/kg of Yreduced significantly compared with the control (P<0.01). See Table14.2. TABLE 14.2 Effects of Extracts X and Y on Water Maze Learning inMice after 6 Days of Administration Blind Blind Blind Blind GroupTerminal 1 Terminal 2 Terminal 3 Terminal 4 Error Frequency EL(sec)control 0.95 ± 0.38 1.09 ± 0.43 0.77 ± 0.61 0.91 ± 0.53 3.82 ± 0.8042.96 ± 13.48 positive 0.73 ± 0.65 1.09 ± 0.30 0.55 ± 0.52 0.82 ± 0.40 3.18 ± 0.75* 32.91 ± 7.15* X I 0.73 ± 0.65 1.27 ± 0.47 0.73 ± 0.65 0.73± 0.65 3.45 ± 1.21 37.18 ± 7.65  X II 0.91 ± 0.30 1.00 ± 0.45 0.64 ±0.92 0.91 ± 0.54 3.45 ± 1.21 37.73 ± 13.26 X III 0.91 ± 0.30 1.09 ± 0.540.91 ± 0.83 0.82 ± 0.75 3.73 ± 1.19  31.09 ± 8.15** Y I 0.91 ± 0.30  1.0± 0.45 0.82 ± 0.40 0.55 ± 0.52 3.36 ± 0.67 35.82 ± 9.93  Y II 0.91 ±0.54 1.09 ± 0.54 1.00 ± 0.77 0.64 ± 0.67 3.64 ± 1.12 35.09 ± 12.13 Y III0.82 ± 0.40 1.09 ± 0.54 0.82 ± 0.60 0.55 ± 0.52 3.27 ± 1.10  31.73 ±8.36***P < 0.05**P < 0.01

4.3. After 9 days of administration of X and Y, the escape latency fromthe water maze by the mice receiving 250 mg/kg of X, 250 mg and 500mg/kg of Y reduced significantly compared with the control (P<0.05). SeeTable 14.3. TABLET 14.3 Effects of Extracts X and Y on Water MazeLearning in Mice after 9 Days of Administration. Blind Blind Blind BlindGroup Terminal 1 Terminal 2 Terminal 3 Terminal 4 Error FrequencyEL(sec) control 0.59 ± 0.73 1.14 ± 0.64 0.55 ± 0.67  1.0 ± 0.76 3.27 ±1.32 39.27 ± 15.52 positive 0.55 ± 0.52 1.00 ± 0.00 0.27 ± 0.65 0.91 ±0.54 2.73 ± 0.65 27.64 ± 6.96* X I 0.45 ± 0.52 1.27 ± 0.47 0.73 ± 0.650.55 ± 0.52 3.00 ± 0.77 33.55 ± 9.59  X II 0.45 ± 0.52 0.91 ± 0.70 0.55± 0.69 0.82 ± 0.89 2.73 ± 0.90 28.00 ± 9.53* X III 0.45 ± 0.52 1.09 ±0.70 0.82 ± 0.75 0.45 ± 0.52 2.82 ± 1.25 29.45 ± 8.49  Y I 0.91 ± 0.700.91 ± 0.54 0.45 ± 0.52 0.55 ± 0.52 2.82 ± 0.98 32.00 ± 9.49  Y II 0.64± 0.50 0.82 ± 0.75 0.64 ± 0.67 0.82 ± 0.60 2.91 ± 1.30 26.36 ± 9.82* YIII 0.73 ± 1.01 0.91 ± 0.30 0.45 ± 0.69 0.55 ± 0.69 2.64 ± 1.12 28.09 ±9.26**P < 0.05

14.4. After 10 days of administration of X and Y, the error frequencymade in the water maze by the mice receiving 250 mg and 500 mg/kg of Y,the escape latency from the water maze by the mice receiving 500 mg/kgof Y reduced significantly compared with the control (P<0.05, P<0.01).See Table 14.4. TABLE 14.4 Effects of Extracts X and Y on Water MazeLearning in Mice after 10 Days of Administration Blind Blind Blind BlindGroup Terminal 1 Terminal 2 Terminal 3 Terminal 4 Error FrequencyEL(sec) control 0.64 ± 0.73 1.14 ± 0.56 0.64 ± 0.66 0.91 ± 0.68 3.32 ±0.95 36.73 ± 13.02 positive 0.45 ± 0.69 0.82 ± 0.40 0.55 ± 0.52 0.82 ±0.40  2.64 ± 0.81*  29.0 ± 10.10 X I 0.36 ± 0.67 0.91 ± 0.70 0.91 ± 0.830.64 ± 0.81 2.82 ± 1.08 33.09 ± 13.96 X II 0.73 ± 0.79 0.82 ± 0.40 0.55± 0.69 0.73 ± 0.47 2.82 ± 0.75 28.91 ± 13.34 X III 0.91 ± 0.70 0.91 ±0.54 0.73 ± 0.65 0.36 ± 0.67 2.91 ± 0.94 32.45 ± 13.57 Y I 0.73 ± 0.790.91 ± 0.30 0.36 ± 0.50 0.73 ± 0.65 2.73 ± 0.90 29.55 ± 13.87 Y II 0.55± 0.52 0.64 ± 0.50 0.45 ± 0.52 0.64 ± 0.67  2.27 ± 0.79** 30.36 ± 12.30Y III 0.45 ± 0.69 1.09 ± 0.54 0.27 ± 0.65 0.55 ± 0.52  2.36 ± 1.21* 25.64 ± 11.02**P < 0.05**P < 0.01

The results indicated that the extracts X and Y had distinct positiveeffects on improving the learning and retention in mice in a water maze.In addition, the effects increased with the period of receiving theextracts of X and Y prolonged.

14.5. After 10 days of receiving X and Y, the mice were administratedwith Na NO2 after the test. The results of treatment with X and Y toprevent impairments induced by Na NO2 in water maze learning in agingmice showed that error frequency made by the mice receiving 100 mg/kaand 200 mg/kg of X, and Y of all doses reduced significantly (P<0.05).See Table 14.5. It indicated the extracts X and Y had distinct positiveeffects on preventing the impairments induced by Na NO₂. See Table 14.5.TABLE 14.5 Effects of Extract X and Y on preventing Impairments Inducedby Na NO₂ Blind Blind Blind Blind Error Group Terminal 1 Terminal 2Terminal 3 Terminal 4 Frequency EL(sec) Control 0.27 ± 0.47 0.91 ± 0.540.55 ± 0.52 1.09 ± 0.54 2.82 ± 0.75  30.91 ± 12.36 Model 1.18 ± 0.750.91 ± 0.30 0.45 ± 0.52 0.73 ± 0.65 3.27 ± 1.01  36.45 ± 16.89 Positive0.45 ± 0.52 0.91 ± 0.30 0.55 ± 0.52 0.45 ± 0.52 2.36 ± 0.81* 32.00 ±15.83 X I 0.55 ± 0.52 0.82 ± 0.60 0.18 ± 0.40 0.82 ± 0.60 2.36 ± 0.81*29.09 ± 13.80 X II 0.18 ± 0.40 0.82 ± 0.60 0.55 ± 0.52 0.82 ± 0.75 2.36± 0.92* 25.82 ± 10.82 X III 0.45 ± 0.52 0.64 ± 0.50 0.82 ± 0.40 0.91 ±0.70 2.82 ± 1.33  31.09 ± 11.76 Y I 0.27 ± 0.47 0.91 ± 0.30 0.36 ± 0.500.73 ± 0.47 2.27 ± 0.79* 27.00 ± 10.73 Y II 0.45 ± 0.52 0.64 ± 0.50 0.36± 0.50 0.91 ± 0.30 2.36 ± 0.81* 25.82 ± 11.43 Y III 0.64 ± 0.50 0.82 ±0.40 0.36 ± 0.50 0.64 ± 0.50 2.45 ± 0.82* 25.09 ± 9.67 *P < 0.05** P < 0.01

Experiment 15: Effects of Wenguanguo (Xanthoceras sorbifolia) Extractson Urination in Mice

As used herein, Extracts (or Fractions) X and Y are different extractsof Wenguanguo plant or Xanthoceras sorbifolia.

Methods of Experiment. One hundred twelve male ICR mice weighing 18-22gm were divided into 8 groups of 14: 1, control: receiving normal saline(NS); 2, DCT group: receiving DCT 33.4 mg/kg; 3, X-I group: receiving100 mg/kg4; 4, X-II group: receiving 200 mg/kg; 5, X-III receiving 400mg/kg; 6, Y-1 group: receiving 125 mg/kg; 7, Y-II group: receiving 250mg/kg and 8, Y-III group: receiving 500 mg/kg.

All the drugs were received with oral administration, 20 ml/kg, once aday for 3 days. After the last administration, the mouse was placed on afilter paper. The filter paper was on the bottom of a 500 ml beak. Thequantity of urine was measured at 30, 60, 120, 180, 240, 300, and 360minutes by weighing the filter paper with the electronic analyticalscale. All data were analyzed with t-test.

Results. After 3 days of administration of X and Y the quantity of urinedischarged at 30 minute by the mice receiving 400 mg/kg of X decreasedsignificantly (P<0.01) compared with the mice receiving normal saline.The quantity of urine discharged at 60 minute by the mice receiving 600mg/kg of Y decreased significantly compared with the mice receivingnormal saline (P<0.01).

The quantity of urine discharged at 180 minutes by the mice receiving200 mg/kg of X, 125 mg and 500 mg/kg of Y decreased significantlycompared with the mice receiving normal saline (P<0.01). See Table 15.1.TABLE 15.1 Effects of Extracts X and Y on the Quantity of Urine in Miceafter 3 Days of Administration Group 30 min 60 min 120 min Control 0.267± 0.105 0.367 ± 0.162 0.382 ± 0.109 Positive 0.348 ± 0.06* 0.471 ±0.169** 0.574 ± 0.249 X I 0.304 ± 0.072 0.274 ± 0.076 0.323 ± 0.173 X II0.341 ± 0.107 0.323 ± 0.102 0.404 ± 0.138 X III 0.155 ± 0.056** 0.200 ±0.140** 0.455 ± 0.211 Y I 0.216 ± 0.130 0.309 ± 0.093 0.341 ± 0.061 Y II0.278 ± 0.063 0.278 ± 0.119 0.437 ± 0.112 Y III 0.227 ± 0.058 0.235 ±0.035** 0.425 ± 0.133Compared with the control group:P < 0.05*P < 0.01**

TABLE 15.1 Effects of Extracts X and Y on the Quantity of Urine in MiceAfter 3 Days of Administration (continued) Group 180 min 240 min 300 min360 min Control 0.191 ± 0.080 0.161 ± 0.083 0.116 ± 0.06  0.103 ± 0.057Positive 0.272 ± 0.131 0.182 ± 0.096 0.110 ± 0.051 0.085 ± 0.031 X I0.184 ± 0.105 0.154 ± 0.093 0.124 ± 0.091 0.102 ± 0.064 X II  0.336 ±0.103** 0.163 ± 0.10  0.107 ± 0.076 0.106 ± 0.075 X III 0.207 ± 0.1120.204 ± 0.088 0.150 ± 0.066 0.116 ± 0.077 Y I  0.367 ± 0.104** 0.171 ±0.085  0.173 ± 0.068* 0.093 ± 0.053 Y II 0.275 ± 0.206 0.145 ± 0.0290.109 ± 0.036 0.106 ± 0.045 Y III  0.319 ± 0.086**  0.264 ± 0.114**0.152 ± 0.084 0.135 ± 0.051Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantityof urine after 3 days of administration of X and Y.

After 5 days of administration of X and Y, the quantity of urinedischarged at 30 minute by the mice receiving 400 mg/kg of X and 500mg/kg of Y decreased, but not significantly compared with the micereceiving normal saline. The quantity of urine discharged at 4 hour bythe mice receiving 400 mg/kg of X and Y in all doses increasedsignificantly compared with the mice receiving normal saline (P<0.05,P<0.01). See Table 15.2. TABLE 15.2 Effects of Extracts X and Y on theQuantity of Urine in Mice after 5 Days of Administration Group 30 min 60min 120 min Control 0.327 ± 0.148 0.330 ± 0.194 0.291 ± 0.146 Positive 0.524 ± 0.206**  0.478 ± 0.185*  0.472 ± 0.292* X I 0.382 ± 0.138 0.251± 0.071 0.265 ± 0.172 X II 0.348 ± 0.144 0.324 ± 0.113 0.277 ± 0.131 XIII 0.245 ± 0.167 0.236 ± 0.129 0.251 ± 0.142 Y I 0.331 ± 0.098 0.340 ±0.133 0.291 ± 0.081 Y II 0.357 ± 0.130 0.290 ± 0.145 0.327 ± 0.157 Y III0.230 ± 0.121 0.307 ± 0.082 0.363 ± 0.100Compared with the control P < 0.05* and P < 0.01**

TABLE 15.2 Effects of Extracts X and Y on the Quantity of Urine in MiceAfter 5 Days of Administration (continued) Group 180 min 240 min 300 min360 min Control 0.186 ± 0.086 0.117 ± 0.069 0.105 ± 0.06  0.104 ± 0.08 Positive 0.214 ± 0.151 0.110 ± 0.045 0.126 ± 0.056 0.112 ± 0.065 X I0.188 ± 0.097 0.175 ± 0.088  0.177 ± 0.102* 0.133 ± 0.092 X II 0.258 ±0.143 0.150 ± 0.077  0.167 ± 0.097* 0.130 ± 0.094 X III 0.226 ± 0.107 0.233 ± 0.132** 0.120 ± 0.059 0.125 ± 0.048 Y I 0.273 ± 0.156  0.215 ±0.095** 0.166 ± 0.151 0.116 ± 0.068 Y II 0.181 ± 0.088  0.181 ± 0.089*0.151 ± 0.104 0.101 ± 0.042 Y III 0.193 ± 0.09   0.217 ± 0.092** 0.112 ±0.056 0.117 ± 0.043Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantityof urine after 5 days of administration of X and Y.

After 7 days of administration of X and Y, the quantity of urine wasmeasured at 30, 60, 120, 180, 240, 300, and 360 minutes. The quantity ofurine discharged at 30 minute by the mice receiving 200, 400 mg/kg of Xand 250, 500 mg/kg of Y decreased significantly (P<0.05) but increasedat 240 minutes compared with the mice receiving normal saline. See Table15.3. TABLE 15.3 Effects of Extracts X and Y on the Quantity of Urine inMice after 7 Days of Administration Group 30 min 60 min 120 min Control0.252 ± 0.142 0.347 ± 0.159 0.430 ± 0.192 Positive  0.434 ± 0.230* 0.606 ± 0.214** 0.590 ± 0.333 X I 0.301 ± 0.152 0.314 ± 0.149 0.342 ±0.186 X II 0.291 ± 0.161 0.332 ± 0.135  0.285 ± 0.173* X III 0.212 ±0.113 0.260 ± 0.103  0.309 ± 0.117* Y I 0.254 ± 0.175 0.283 ± 0.1370.313 ± 0.178 Y II 0.261 ± 0.189 0.292 ± 0.129  0.300 ± 0.128* Y III0.246 ± 0.170 0.268 ± 0.240  0.281 ± 0.146*Compared with the control P < 0.05* and P < 0.01**

TABLE 15.3 Effects of Extracts X and Y on the Quantity of Urine in Miceafter 7 Days of Administration (continued) Group 180 min 240 min 300 min360 min Control 0.285 ± 0.136 0.155 ± 0.119 0.122 ± 0.071 0.111 ± 0.061Positive 0.314 ± 0.119  0.279 ± 0.192* 0.163 ± 0.087 0.148 ± 0.071 X I0.267 ± 0.179 0.200 ± 0.114 0.176 ± 0.147 0.157 ± 0.077 X II 0.250 ±0.116 0.203 ± 0.134  0.180 ± 0.079* 0.129 ± 0.085 X III 0.293 ± 0.142 0.250 ± 0.116*  0.194 ± 0.104* 0.151 ± 0.076 Y I 0.310 ± 0.168 0.248 ±0.178 0.155 ± 0.108 0.113 ± 0.05  Y II 0.334 ± 0.208 0.259 ± 0.205 0.205 ± 0.109*  0.188 ± 0.113* Y III 0.267 ± 0.133 0.212 ± 0.125  0.205± 0.119*  0.169 ± 0.073*Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantityof urine after 7 days of administration of X and Y.

After 10 days of administration of X and Y, the quantity of urinedischarged at 120 minutes by the mice receiving 200, 400 mg/kg of X and250, 500 mg/kg of Y decreased significantly (P<0.05) compared with themice receiving normal saline. See FIG. 59. TABLE 15.4 Effects ofExtracts X and Y on the Quantity of Urine in Mice after 10 Days ofAdministration Group 30 min 60 min 120 min Control 0.292 ± 0.184 0.323 ±0.158 0.418 ± 0.221 Positive 0.374 ± 0.159  0.432 ± 0.163*  0.643 ±0.181** X I 0.306 ± 0.124 0.317 ± 0.088 0.339 ± 0.145 X II 0.292 ± 0.0820.343 ± 0.120  0.279 ± 0.118* X III 0.266 ± 0.116 0.348 ± 0.161  0.274 ±0.111* Y I 0.273 ± 0.117 0.331 ± 0.103 0.406 ± 0.175 Y II 0.289 ± 0.1260.344 ± 0.147  0.254 ± 0.102* Y III 0.227 ± 0.129 0.322 ± 0.162  0.255 ±0.124*Compared with the control P < 0.05* and P < 0.01**

TABLE 15.4 Effects of Extracts X and Y on the Quantity of Urine in Miceafter 10 Days of Administration (continued) Group 180 min 240 min 300min 360 min Control 0.203 ± 0.087 0.144 ± 0.098 0.108 ± 0.074 0.091 ±0.060 Positive 0.253 ± 0.116 0.147 ± 0.067 0.095 ± 0.094 0.068 ± 0.049 XI 0.249 ± 0.094 0.172 ± 0.093 0.120 ± 0.058 0.093 ± 0.050 X II 0.225 ±0.074 0.163 ± 0.051 0.116 ± 0.052 0.093 ± 0.051 X III 0.247 ± 0.1040.186 ± 0.102 0.121 ± 0.053 0.098 ± 0.065 Y I 0.243 ± 0.101 0.171 ±0.098 0.126 ± 0.086 0.098 ± 0.058 Y II 0.229 ± 0.097 0.164 ± 0.091 0.124± 0.094 0.111 ± 0.067 Y III 0.213 ± 0.102 0.170 ± 0.081 0.121 ± 0.0590.095 ± 0.045Compared with the control P < 0.05* and P < 0.01**

The results indicated that the extract X and Y can regulate the quantityof urine after 10 days of administration of X and Y.

Conclusion. The results indicated that the extract X and Y can regulatethe quantity of urine after 3-10 days of administration of X and Y.

Experiment 15A: Antidiuresis Test of Rat in Metabolism Cage

The antidiuresis tests using Xanthoceras Sorbifolia Extract FS(X) andXanthoceras Sorbifolia saponin extract FS(Y) were carried out in rats.Note: Extracts FS(X) and FS(Y) similar to Extracts X and Y respectively.FS(Y) and FS(X) are crude saponin extracts and the “FS(Y)” and “FS(X)”are the extract names that were used in the experiments.

SD rats were treated with FS(X) by oral administration, daily at thedoses of 100, 200, 400 mg/kg for 25 days. The experiment included anegative control group and positive control. Collecting urine inStandard metabolism cage and observation indicators, including urinevolume, urine Na+, K+, Cl− content, pH and osmotic pressure. The resultsobtained were as follows: (1) Extract FS(X) has dose-dependantantidiuresis effect. There is significant antidiuresis at all the timephases for 200 and 400 mg/kg. However, by urination rate, the 400 mg/kgdosage group has a more significant effect in that it can delayurination output by two hours. In addition, there is no change in theimpact on total urine volume output. (2) The extract FS(X) increased theconcentration of ions in urine after drug treatment. However, there isno apparent dose dependency. (3)FS (X) and FS (Y) have no significantimpact on the pH value and osmotic pressure, but have slight impact onthe specific gravity.

Objective. Standard metabolism cage method was carried out in rats toresearch the antidiuresis of FS(X) and FS(Y).

Test Drug. FS(X) is compositions of Xanthoceras Sorbifolia extract.FS(Y) is compositions of Xanthoceras Sorbifolia extract. The test drugis a suspension formulated with 0.5% sodium carboxymethyl cellulose(0.5% CMC—Na).

Test Animals. Male SD rats, initial weight 150˜200 g, 100 individuals.The test animals are raised in cages (volume: 20×30×45 cm), and eachcage has 5 animals. The basal feed is the full-rate pellet feed forexperimental rat, self-made by the Experimental Animal Center. The cagebottom padding is wood shaving and chaff, dried before use. After thepadding replacement that is made every other day on average, the cagesare disinfected before reuse. Laboratory temperature 23±2° C., humidity40˜70%, with air-conditioning, exhaust and ventilation equipment,natural lighting and a light-shade cycle of about 12 hours.

Animal Screening. The animals are placed individually in the cage foradaptation once a day for 6˜10 hours per day for 2 days. Before placingthem into the case, press the lower abdomen gently to discharge theremaining urine, inject 38° C. distilled water into the stomach at thevolume of 25 ml/kg as the water load. Collect the urine in themetabolism cage within 2 hours after stomach injection; collect theremaining urine by pressing the lower abdomen gently immediately beforetaking them out of the cage. Animals whose urine volume attains above40% of the injection volume will be qualified ones.

Test Instruments. Standard metabolism cage, Automatic urine analyzer(Miditron Junior II), Urine osmotic pressure tester, and Urine iontester (EL-ISE, Beckman).

Methods

Test groups. There are 3 groups for the FS (X), i.e., for 100, 200, 400mg/kg/day, respectively. There is a dosage group for FS (Y): 400mg/kg/day; once a day, fed at 30 min after administration, for 25 days.Control group were fed with 0.5% CMC—Na into the stomach every day. Thepositive control drug is pituitrin, injected into the abdominal cavityat the rate of 0.25 u/kg before the animals are placed in the cage. Eachgroup includes 10 animals.

Urine collection and observation indicators. For the test groups, theanimals that have experienced the administration period are on diet for18 hours before urine collection without prohibiting drinking. Afterpressing their lower abdomen gently to discharge the remaining urine,apply the water load of 38° C. distilled water at the volume of 50ml/kg, and place them in the cage for urine collection. Collect theurine volume and times at 0.25 hr, 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hrand 6 hr after water feeding, when the animals are taken out of the cageat 6 hr, press their lower abdomen gently to collect the remaining urinein the bladder. Conduct the routine urine examination (pH, erythrocyte,leucocyte, protein, etc); measure urine Na+, K+, Cl− concentrations andurine osmotic pressure for the urine samples.

Data processing. Urination speed, Relative urine volume, Urine Na+, K+,Cl− content data are shown in X±Sd, and are compared with the controlgroup and subject to a student t-test.

Results

Impact on urine volume. FS (X) has dose-dependant antidiuresis effects.There is a significant antidiuresis effect at all the time phases usingdosages of 200 and 400 mg/kg. See Table 15A-1, FIGS. 63 and 63A.However, by urination discharge rate, the 400 mg/kg group has a moresignificant effect in reducing the urine output during the first 2 hrsas compared with the control. See Table 15A-2, FIGS. 64 and 64A. The FS(Y) 400 mg/kg group has an equivalent drug efficacy to FX (X) 400 mg/kgduring the first 2 hours of experiment. In addition, there is noapparent dose dependency in the impact on total urine volume during thewhole experiment. The 400 mg/kg groups of FS(X) can significantly reducethe urine volume during the first 6 hours after the drug-treatment whichis more effective than the positive control drug (pituitrin).

Impact on Na+, K+, Cl− ions in urine. By reducing the urine volume, thetest drug has increased the concentration of ions in urine to differentextent. However, there is no apparent dose dependency. By ionic, thereis almost no impact on Na+ and Cl− ions, there is a certain dischargepromotion action on K+ ions. The concentration and discharge amount ofall ions in urine from the positive drug have risen apparently.

Impact on urine pH and osmotic pressure. FS (X) and FS (Y) have nosignificant impact on the pH value and osmotic pressure, but have slightimpact on the specific gravity.

FIGS. 63 and 63A, Table 15A-1, shows results of the urine volume withwater load after FS(X) Xanthoceras Sorbifolia Extract administration for25 days. FIGS. 64 and 64A, Table 15A-2, shows results of dischargingurine speed with water load after FS(X) Xanthoceras Sorbifolia Extractadministration for 25 days. FIG. 65, Table 15A-3, shows results of urinespecific gravity and pH with water load after FS(X) XanthocerasSorbifolia Extract administration for 25 days. FIG. 66, Table 15A-4,Concentration of Na+, K+ and Cl− in urine with water load after FS(X)Xanthoceras Sorbifolia Extract administration for 25 days.

Although the present invention has been described in detail withparticular reference to preferred embodiments thereof, it should beunderstood that the invention is capable of other different embodiments,and its details are capable of modifications in various obvious aspects.As is readily apparent to those skilled in the art, variations andmodifications can be affected while remaining within the spirit andscope of the invention. Accordingly, the foregoing disclosure,description, and figures are for illustrative purpose only, and do notin any way limit the invention which is defined only by the claims.TABLE 5.2 Chemical Shift Data of 2D NMR chemical shift of HMQC analysisof compound Y DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO= 5, PROCNO = 1 F1PLO = 144.360 ppm, F1PHI = 10.797 ppm, F2PLO = 7.966ppm, F2PHI = 0.417 ppm MI = 1.00 cm, MAXI = 10000.00 cm, PC = 1.400ADDRESS FREQUENCY row [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1148 20698.986 137.1608 384 3574.377 5.9557 2.00 2 152 20564.195 136.2676401 3471.337 5.7840 2.67 3 157 20465.209 135.6117 220 4533.779 7.554345.24 4 223 18893.424 125.1963 431 3295.261 5.4906 6.22 5 234 18649.311123.5787 258 4311.820 7.1845 100.00 6 315 16736.119 110.9011 3763620.289 6.0322 7.49 7 353 15834.069 104.9237 493 2934.550 4.8896 3.98 8355 15778.398 104.5548 449 3192.387 5.3192 2.99 9 355 15778.398 104.5548492 2936.414 4.8927 1.18 10 451 13524.788 89.6213 660 1951.827 3.25222.41 11 451 13524.788 89.6213 663 1934.401 3.2231 2.60 12 473 12994.27486.1059 563 2520.987 4.2005 1.93 13 479 12861.933 85.2290 500 2891.9334.8186 3.96 14 491 12583.008 83.3807 487 2967.012 4.9437 6.95 15 52311826.204 78.3658 307 4025.464 6.7073 3.92 16 523 11826.204 78.3658 3094011.801 6.6846 4.78 17 523 11826.204 78.3658 545 2627.492 4.3780 1.9918 529 11690.020 77.4633 504 2866.164 4.7757 4.60 19 532 11624.01677.0260 530 2713.960 4.5221 2.32 20 532 11624.016 77.0260 532 2703.5634.5047 2.51 21 535 11536.339 76.4450 590 2363.678 3.9384 3.34 22 54511299.475 74.8754 573 2461.387 4.1012 2.35 23 545 11299.475 74.8754 5762447.179 4.0775 2.10 24 555 11063.554 73.3121 541 2653.693 4.4216 6.6225 567 10795.113 71.5333 537 2673.042 4.4539 1.84 26 567 10795.11371.5333 539 2662.683 4.4366 2.82 27 567 10795.113 71.5333 541 2650.9334.4170 1.72 28 579 10495.725 69.5494 527 2734.037 4.5555 5.06 29 59410156.363 67.3006 563 2523.651 4.2050 7.18 30 622 9486.037 62.8588 6082256.690 3.7601 2.91 31 622 9486.037 62.8588 611 2241.336 3.7346 2.67 32622 9486.037 62.8588 635 2100.199 3.4994 2.96 33 622 9486.037 62.8588637 2086.756 3.4770 3.42 34 627 9381.439 62.1656 552 2586.967 4.31053.58 35 627 9381.439 62.1656 555 2568.334 4.2794 3.42 36 627 9381.43962.1656 568 2494.546 4.1565 2.54 37 627 9381.439 62.1656 571 2474.5594.1232 2.51 38 630 9297.809 61.6115 531 2709.734 4.5150 2.61 39 6309297.809 61.6115 539 2660.734 4.4334 2.66 40 670 8349.676 55.3287 911480.283 0.8003 2.14 41 670 8349.676 55.3287 913 465.738 0.7760 2.18 42726 7038.860 46.6427 679 1839.659 3.0653 2.51 43 726 7038.860 46.6427819 1016.627 1.6939 1.42 44 726 7038.860 46.6427 848 848.511 1.4138 2.4945 764 6151.769 40.7644 679 1841.145 3.0678 6.40 46 764 6151.769 40.7644682 1821.592 3.0352 1.04 47 777 5836.727 38.6768 850 837.869 1.3961 2.1548 777 5836.727 38.6768 853 818.861 1.3644 2.05 49 777 5836.727 38.6768907 500.256 0.8335 1.64 50 791 5512.022 36.5251 775 1277.340 2.1283 2.3851 791 5512.022 36.5251 778 1258.929 2.0977 1.38 52 791 5512.022 36.5251785 1218.892 2.0309 1.38 53 791 5512.022 36.5251 788 1201.847 2.00251.09 54 837 4417.038 29.2693 881 655.973 1.0930 23.74 55 848 4174.67927.6633 864 757.206 1.2617 20.53 56 848 4174.679 27.6633 872 709.0601.1815 3.17 57 856 3984.149 26.4008 774 1281.339 2.1350 1.66 58 8563984.149 26.4008 778 1259.751 2.0990 1.59 59 856 3984.149 26.4008 8041107.062 1.8446 1.02 60 856 3984.149 26.4008 807 1089.219 1.8149 1.21 61873 3578.068 23.7099 799 1138.137 1.8964 1.68 62 873 3578.068 23.7099814 1046.185 1.7432 1.26 63 891 3142.837 20.8259 788 1200.655 2.000613.95 64 891 3142.837 20.8259 804 1107.046 1.8446 28.24 65 894 3086.14720.4502 788 1200.275 1.9999 3.08 66 894 3086.147 20.4502 804 1106.8031.8442 1.17 67 894 3086.147 20.4502 815 1041.758 1.7358 27.19 68 8943086.147 20.4502 858 789.804 1.3160 1.16 69 897 3015.337 19.9810 858790.292 1.3168 33.47 70 906 2802.854 18.5730 830 953.652 1.5890 1.35 71906 2802.854 18.5730 834 931.208 1.5516 1.89 72 914 2613.995 17.3215 892592.663 0.9875 12.71 73 919 2490.082 16.5004 875 693.445 1.1554 8.90 74919 2490.082 16.5004 880 660.343 1.1003 1.76 75 925 2342.840 15.5247 7781258.345 2.0967 5.45 76 925 2342.840 15.5247 782 1237.122 2.0613 5.31 77925 2342.840 15.5247 791 1183.410 1.9718 2.33 78 925 2342.840 15.5247795 1159.732 1.9324 2.38 79 925 2342.840 15.5247 907 503.260 0.8385 6.2780 925 2342.840 15.5247 912 474.699 0.7910 7.15

TABLE 5.3 Chemical Shift Data of 2D NMR chemical shift of HMBC analysisof Y DU = C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO = 6,PROCNO = 1 F1PLO = 178.339 ppm, F1PHI = 10.721 ppm, F2PLO = 6.881 ppm,F2PHI = 0.573 ppm MI = 1.00 cm, MAXI = 10000.00 cm, PC = 1.400 ADDRESSFREQUENCY row [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 12326590.750 176.2058 895 573.276 0.9552 3.11 2 145 25939.373 171.8894 5312710.686 4.5166 2.92 3 145 25939.373 171.8894 539 2662.033 4.4355 2.54 4166 25312.006 167.7321 308 4016.437 6.6923 3.45 5 166 25312.006 167.7321346 3793.847 6.3214 14.90 6 166 25312.006 167.7321 348 3785.322 6.307212.47 7 166 25312.006 167.7321 385 3564.443 5.9392 1.47 8 166 25312.006167.7321 400 3477.948 5.7950 4.16 9 166 25312.006 167.7321 403 3458.5525.7627 3.96 10 166 25312.006 167.7321 781 1243.983 2.0728 1.17 11 16625312.006 167.7321 788 1200.447 2.0002 10.44 12 166 25312.006 167.7321793 1171.992 1.9528 3.35 13 166 25312.006 167.7321 815 1041.336 1.735133.24 14 290 21640.068 143.3997 679 1839.970 3.0658 5.67 15 29021640.068 143.3997 788 1199.609 1.9988 1.03 16 290 21640.068 143.3997804 1107.222 1.8449 33.90 17 290 21640.068 143.3997 813 1053.918 1.75612.30 18 290 21640.068 143.3997 848 848.155 1.4132 1.25 19 322 20697.354137.1527 780 1246.505 2.0770 31.46 20 322 20697.354 137.1527 7881200.075 1.9996 44.60 21 322 20697.354 137.1527 793 1170.377 1.9501 3.7922 322 20697.354 137.1527 815 1040.860 1.7343 4.31 23 327 20566.367136.2847 780 1246.629 2.0772 3.06 24 327 20566.367 136.2847 788 1201.1922.0015 5.78 25 327 20566.367 136.2847 793 1170.748 1.9507 47.25 26 32720566.367 136.2847 815 1041.067 1.7346 58.19 27 365 19434.006 128.7811780 1245.861 2.0759 16.02 28 365 19434.006 128.7811 788 1200.442 2.000233.73 29 365 19434.006 128.7811 793 1171.204 1.9515 46.70 30 36519434.006 128.7811 815 1040.940 1.7344 80.60 31 384 18893.113 125.1968679 1839.547 3.0651 9.80 32 384 18893.113 125.1968 788 1200.729 2.00072.34 33 384 18893.113 125.1968 801 1124.942 1.8744 1.93 34 384 18893.113125.1968 803 1113.960 1.8561 1.36 35 384 18893.113 125.1968 809 1077.5561.7954 3.32 36 384 18893.113 125.1968 813 1051.694 1.7524 3.14 37 38418893.113 125.1968 848 847.499 1.4121 1.45 38 457 16738.236 110.9173 3613707.331 6.1772 6.44 39 457 16738.236 110.9173 390 3534.169 5.8887 6.7540 457 16738.236 110.9173 486 2972.803 4.9534 1.24 41 457 16738.236110.9173 488 2962.223 4.9357 1.40 42 457 16738.236 110.9173 563 2520.5594.1998 12.21 43 488 15822.760 104.8508 531 2712.055 4.5189 1.39 44 48815822.760 104.8508 538 2668.887 4.4470 4.68 45 488 15822.760 104.8508545 2627.709 4.3783 14.61 46 488 15822.760 104.8508 660 1952.474 3.25332.34 47 488 15822.760 104.8508 662 1941.101 3.2343 3.52 48 488 15822.760104.8508 664 1928.930 3.2140 2.41 49 489 15777.470 104.5507 538 2669.1014.4473 12.69 50 489 15777.470 104.5507 546 2622.941 4.3704 6.06 51 48915777.470 104.5507 590 2363.695 3.9384 3.08 52 489 15777.470 104.5507660 1953.443 3.2549 2.02 53 489 15777.470 104.5507 662 1940.913 3.23401.47 54 566 13527.920 89.6439 492 2938.198 4.8957 11.15 55 566 13527.92089.6439 805 1101.852 1.8359 1.93 56 566 13527.920 89.6439 851 830.5521.3839 5.09 57 566 13527.920 89.6439 864 757.374 1.2620 53.64 58 56613527.920 89.6439 874 696.110 1.1599 39.08 59 566 13527.920 89.6439 880658.836 1.0978 4.62 60 566 13527.920 89.6439 911 480.432 0.8005 1.38 61566 13527.920 89.6439 913 466.506 0.7773 1.40 62 584 12992.842 86.0981376 3621.487 6.0342 5.29 63 584 12992.842 86.0981 531 2708.564 4.51311.17 64 584 12992.842 86.0981 539 2662.648 4.4366 4.18 65 584 12992.84286.0981 545 2625.947 4.3754 6.92 66 584 12992.842 86.0981 864 757.0401.2614 1.36 67 588 12860.941 85.2241 370 3655.044 6.0901 2.76 68 58812860.941 85.2241 376 3621.609 6.0344 10.88 69 588 12860.941 85.2241 5542574.294 4.2893 1.21 70 588 12860.941 85.2241 864 756.227 1.2600 1.05 71598 12585.614 83.3996 376 3621.268 6.0338 1.92 72 598 12585.614 83.3996475 3038.901 5.0635 1.23 73 598 12585.614 83.3996 500 2890.984 4.81701.60 74 598 12585.614 83.3996 505 2863.233 4.7708 3.39 75 598 12585.61483.3996 881 655.156 1.0916 1.11 76 623 11822.784 78.3447 295 4093.8186.8212 2.30 77 623 11822.784 78.3447 321 3940.803 6.5663 2.25 78 62311822.784 78.3447 348 3785.120 6.3069 18.28 79 623 11822.784 78.3447 4343277.040 5.4603 1.13 80 623 11822.784 78.3447 448 3194.918 5.3234 7.2681 623 11822.784 78.3447 563 2521.171 4.2008 7.36 82 623 11822.78478.3447 845 867.271 1.4451 1.26 83 623 11822.784 78.3447 849 844.6691.4074 10.09 84 623 11822.784 78.3447 858 790.448 1.3171 26.63 85 62311822.784 78.3447 881 656.434 1.0938 51.16 86 630 11619.674 76.9987 3483785.085 6.3068 1.21 87 630 11619.674 76.9987 539 2661.936 4.4354 6.4988 630 11619.674 76.9987 552 2584.607 4.3065 1.49 89 630 11619.67476.9987 858 790.978 1.3179 2.74 90 630 11619.674 76.9987 881 655.9771.0930 5.21 91 641 11304.499 74.9102 307 4021.328 6.7004 1.03 92 64111304.499 74.9102 526 2736.013 4.5588 4.14 93 641 11304.499 74.9102 5382670.608 4.4498 9.80 94 641 11304.499 74.9102 881 656.537 1.0939 1.61 95650 11028.925 73.0841 308 4015.948 6.6915 16.79 96 650 11028.925 73.0841346 3793.459 6.3207 13.94 97 650 11028.925 73.0841 526 2736.761 4.56017.46 98 650 11028.925 73.0841 574 2454.410 4.0896 4.70 99 650 11028.92573.0841 610 2245.856 3.7421 11.08 100 650 11028.925 73.0841 637 2088.8953.4806 5.03 101 650 11028.925 73.0841 679 1839.510 3.0650 8.83 102 65011028.925 73.0841 848 850.226 1.4167 1.52 103 650 11028.925 73.0841 881656.986 1.0947 1.80 104 658 10790.329 71.5030 308 4016.477 6.6923 1.26105 658 10790.329 71.5030 531 2708.840 4.5135 2.58 106 658 10790.32971.5030 564 2518.203 4.1959 5.49 107 658 10790.329 71.5030 609 2253.8413.7554 1.07 108 658 10790.329 71.5030 881 655.677 1.0925 1.26 109 66810496.015 69.5527 530 2715.370 4.5244 1.73 110 668 10496.015 69.5527 5332699.760 4.4984 1.85 111 668 10496.015 69.5527 540 2658.708 4.4300 3.19112 668 10496.015 69.5527 590 2363.392 3.9379 4.52 113 668 10496.01569.5527 804 1107.602 1.8455 1.00 114 668 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431 3294.477 5.4893 5.30 135 781 7163.191 47.4675 539 2661.4764.4346 3.10 136 781 7163.191 47.4675 541 2647.966 4.4121 3.42 137 7817163.191 47.4675 564 2517.984 4.1955 1.14 138 781 7163.191 47.4675 6791839.442 3.0649 3.64 139 781 7163.191 47.4675 804 1107.164 1.8448 37.94140 781 7163.191 47.4675 822 1002.093 1.6697 1.62 141 781 7163.19147.4675 849 845.058 1.4081 2.98 142 781 7163.191 47.4675 858 790.2031.3167 4.12 143 781 7163.191 47.4675 874 695.441 1.1588 1.07 144 7817163.191 47.4675 881 656.256 1.0935 6.01 145 781 7163.191 47.4675 891594.695 0.9909 39.60 146 781 7163.191 47.4675 909 489.613 0.8158 3.90147 786 7041.051 46.6581 432 3290.762 5.4831 2.39 148 786 7041.05146.6581 669 1898.191 3.1628 1.21 149 786 7041.051 46.6581 677 1850.6533.0836 2.37 150 786 7041.051 46.6581 680 1835.948 3.0591 2.84 151 7867041.051 46.6581 682 1821.518 3.0351 2.66 152 786 7041.051 46.6581 6891782.432 2.9699 2.02 153 786 7041.051 46.6581 776 1274.214 2.1231 2.05154 786 7041.051 46.6581 800 1129.576 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834 5623.89637.2672 858 790.603 1.3173 4.03 217 834 5623.896 37.2672 864 756.7681.2609 1.89 218 834 5623.896 37.2672 874 696.444 1.1604 2.15 219 8345623.896 37.2672 881 656.011 1.0931 4.39 220 834 5623.896 37.2672 891594.776 0.9910 4.19 221 834 5623.896 37.2672 909 489.272 0.8152 10.53222 837 5533.835 36.6704 777 1268.681 2.1139 1.72 223 837 5533.83536.6704 804 1108.282 1.8466 1.01 224 837 5533.835 36.6704 811 1067.1201.7781 1.70 225 837 5533.835 36.6704 819 1017.407 1.6952 4.37 226 8375533.835 36.6704 822 1001.730 1.6691 4.67 227 837 5533.835 36.6704 832943.762 1.5725 2.78 228 837 5533.835 36.6704 849 841.934 1.4028 2.09 229837 5533.835 36.6704 858 790.630 1.3174 8.57 230 837 5533.835 36.6704864 757.963 1.2629 2.10 231 837 5533.835 36.6704 874 695.245 1.1584 1.83232 837 5533.835 36.6704 881 656.351 1.0936 11.03 233 837 5533.83536.6704 891 594.692 0.9909 25.74 234 837 5533.835 36.6704 909 489.1390.8150 53.53 235 837 5533.835 36.6704 913 467.893 0.7796 6.55 236 8405450.419 36.1177 307 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257 8754418.238 29.2778 882 648.470 1.0805 1.77 258 875 4418.238 29.2778 892593.301 0.9886 19.66 259 883 4176.082 27.6732 662 1941.300 3.2346 1.71260 883 4176.082 27.6732 853 819.336 1.3652 15.64 261 883 4176.08227.6732 857 794.801 1.3243 4.59 262 883 4176.082 27.6732 863 763.3741.2720 1.70 263 883 4176.082 27.6732 874 696.058 1.1598 89.76 264 8834176.082 27.6732 878 671.861 1.1195 2.96 265 883 4176.082 27.6732 881657.198 1.0950 7.96 266 883 4176.082 27.6732 911 479.487 0.7989 2.54 267883 4176.082 27.6732 913 466.645 0.7775 2.27 268 903 3588.775 23.7813431 3296.117 5.4921 2.85 269 903 3588.775 23.7813 791 1182.756 1.97071.05 270 903 3588.775 23.7813 801 1127.886 1.8793 1.45 271 903 3588.77523.7813 804 1108.540 1.8471 1.43 272 903 3588.775 23.7813 811 1064.1281.7731 1.37 273 903 3588.775 23.7813 821 1006.100 1.6764 3.82 274 9033588.775 23.7813 874 696.649 1.1608 2.42 275 903 3588.775 23.7813 881655.757 1.0926 2.43 276 917 3174.632 21.0370 307 4021.419 6.7006 1.44277 917 3174.632 21.0370 384 3571.435 5.9508 2.36 278 917 3174.63221.0370 403 3463.331 5.7707 1.11 279 917 3174.632 21.0370 561 2531.8504.2186 5.21 280 917 3174.632 21.0370 564 2517.700 4.1950 5.22 281 9173174.632 21.0370 778 1262.748 2.1040 3.52 282 917 3174.632 21.0370 7931170.055 1.9496 22.51 283 917 3174.632 21.0370 800 1133.275 1.8883 2.77284 917 3174.632 21.0370 805 1100.419 1.8335 3.08 285 917 3174.63221.0370 809 1079.427 1.7986 2.91 286 917 3174.632 21.0370 815 1043.1181.7381 24.13 287 917 3174.632 21.0370 827 975.020 1.6246 2.05 288 9173174.632 21.0370 848 851.242 1.4184 1.60 289 917 3174.632 21.0370 864757.267 1.2618 1.67 290 917 3174.632 21.0370 869 727.306 1.2119 1.80 291917 3174.632 21.0370 881 656.407 1.0937 6.11 292 922 3013.427 19.9687309 4014.743 6.6895 13.55 293 922 3013.427 19.9687 401 3472.374 5.78572.11 294 922 3013.427 19.9687 679 1841.875 3.0690 7.26 295 922 3013.42719.9687 777 1263.952 2.1060 1.96 296 922 3013.427 19.9687 794 1165.7251.9424 1.57 297 922 3013.427 19.9687 799 1137.940 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2611.793 17.3073 822 1001.860 1.6693 7.01 319 936 2611.79317.3073 864 757.418 1.2620 6.44 320 936 2611.793 17.3073 869 725.7531.2093 1.48 321 936 2611.793 17.3073 872 707.264 1.1785 1.56 322 9362611.793 17.3073 881 654.782 1.0910 17.83 323 936 2611.793 17.3073 902531.793 0.8861 19.86 324 936 2611.793 17.3073 909 490.194 0.8168 2.94325 936 2611.793 17.3073 920 427.649 0.7126 1.52 326 940 2489.58616.4974 662 1941.819 3.2355 2.46 327 940 2489.586 16.4974 664 1929.1723.2144 1.76 328 940 2489.586 16.4974 822 1001.010 1.6679 1.20 329 9402489.586 16.4974 864 757.021 1.2614 66.00 330 940 2489.586 16.4974 871712.622 1.1874 10.06 331 940 2489.586 16.4974 880 660.618 1.1007 2.75332 940 2489.586 16.4974 885 632.387 1.0537 18.76 333 940 2489.58616.4974 891 594.244 0.9901 3.19 334 940 2489.586 16.4974 899 548.1420.9133 1.83 335 940 2489.586 16.4974 911 478.615 0.7975 8.03 336 9402489.586 16.4974 913 466.429 0.7772 10.10 337 940 2489.586 16.4974 920426.462 0.7106 1.57 338 945 2348.374 15.5617 383 3576.612 5.9594 2.25339 945 2348.374 15.5617 402 3464.839 5.7732 1.77 340 945 2348.37415.5617 769 1311.156 2.1847 9.92 341 945 2348.374 15.5617 783 1233.2082.0548 4.90 342 945 2348.374 15.5617 791 1184.039 1.9729 8.41 343 9452348.374 15.5617 804 1106.429 1.8436 5.06 344 945 2348.374 15.5617 8191018.365 1.6968 3.91 345 945 2348.374 15.5617 822 1001.834 1.6693 5.15346 945 2348.374 15.5617 864 756.741 1.2609 6.60 347 945 2348.37415.5617 872 710.417 1.1837 1.20 348 945 2348.374 15.5617 881 655.0391.0914 2.79 349 945 2348.374 15.5617 885 632.486 1.0539 2.24 350 9452348.374 15.5617 899 550.147 0.9167 19.61 351 945 2348.374 15.5617 906510.518 0.8506 3.78 352 945 2348.374 15.5617 911 480.301 0.8003 8.74 353945 2348.374 15.5617 914 464.362 0.7737 8.49 354 945 2348.374 15.5617920 426.538 0.7107 18.76

TABLE 5.5 Chemical Shift of the Proton NMR of compound Y of XanthocerasSorbifolia Extract FREQUENCY # ADDRESS [Hz] [PPM] INTENSITY 1 5031.95309.524 8.8468 0.46 2 5430.0 5222.264 8.7015 134.66 3 5840.9 5132.1738.5513 0.48 4 8204.9 4613.901 7.6878 0.26 5 8568.1 4534.263 7.5551 88.286 8943.7 4451.919 7.4179 0.33 7 9209.7 4393.602 7.3207 0.80 8 9575.24313.488 7.1872 180.00 9 9952.1 4230.846 7.0495 0.87 10 10277.2 4159.5856.9308 1.02 11 10886.8 4025.922 6.7081 5.68 12 10933.6 4015.677 6.69106.19 13 11939.8 3795.084 6.3235 6.26 14 11986.7 3784.803 6.3063 5.85 1512576.0 3655.587 6.0910 1.47 16 12728.0 3622.280 6.0355 6.92 17 12880.53588.844 5.9798 1.87 18 12914.2 3581.450 5.9675 4.48 19 12946.5 3574.3665.9557 4.59 20 12979.5 3567.130 5.9436 2.00 21 13382.6 3478.754 5.79642.41 22 13415.6 3471.539 5.7844 5.03 23 13447.3 3464.568 5.7727 5.15 2413479.1 3457.598 5.7611 2.66 25 14218.8 3295.432 5.4909 14.00 26 14655.93199.603 5.3312 28.97 27 14691.2 3191.875 5.3184 27.63 28 15715.32967.359 4.9443 5.64 29 15826.3 2943.028 4.9037 4.82 30 15860.6 2935.5044.8912 5.15 31 16047.0 2894.632 4.8231 4.20 32 16149.6 2872.131 4.78563.79 33 16171.9 2867.242 4.7775 4.42 34 16774.0 2735.249 4.5575 5.92 3516856.4 2717.187 4.5274 4.26 36 16883.5 2711.235 4.5175 4.56 37 16903.52706.854 4.5102 5.89 38 16930.6 2700.914 4.5003 4.68 39 17021.2 2681.0464.4672 2.99 40 17056.9 2673.228 4.4542 8.41 41 17099.1 2663.974 4.43888.87 42 17135.6 2655.976 4.4254 8.11 43 17200.4 2641.771 4.4018 1.88 4417232.6 2634.706 4.3900 2.90 45 17271.0 2626.288 4.3760 4.33 46 17308.82618.001 4.3622 2.85 47 17401.8 2597.607 4.3282 0.83 48 17459.1 2585.0434.3073 3.41 49 17512.6 2573.328 4.2877 4.04 50 17596.4 2554.947 4.25710.89 51 17628.2 2547.968 4.2455 0.80 52 17666.8 2539.518 4.2314 1.30 5317723.0 2527.184 4.2109 6.71 54 17745.9 2522.172 4.2025 7.98 55 17791.22512.248 4.1860 2.28 56 17874.1 2494.071 4.1557 2.90 57 17895.0 2489.4774.1480 3.09 58 17927.1 2482.454 4.1363 2.64 59 17948.8 2477.683 4.12842.51 60 18034.1 2458.993 4.0972 3.27 61 18064.6 2452.313 4.0861 2.96 6218076.5 2449.702 4.0817 2.96 63 18118.3 2440.538 4.0665 0.72 64 18156.42432.180 4.0526 0.89 65 18196.3 2423.432 4.0380 0.44 66 18432.1 2371.7353.9518 2.34 67 18459.7 2365.684 3.9418 4.30 68 18487.4 2359.600 3.93162.24 69 18962.4 2255.466 3.7581 3.86 70 19011.0 2244.816 3.7404 4.38 7119669.3 2100.485 3.4999 4.39 72 19717.6 2089.898 3.4822 4.16 73 20365.21947.920 3.2457 2.42 74 20401.1 1940.054 3.2326 2.35 75 20418.9 1936.1613.2261 2.38 76 20751.3 1863.290 3.1047 0.70 77 20815.8 1849.152 3.08113.20 78 20857.9 1839.919 3.0657 11.24 79 20924.8 1825.236 3.0412 1.13 8023431.3 1275.730 2.1257 3.95 81 23491.3 1262.573 2.1037 5.36 82 23538.71252.187 2.0864 15.56 83 23570.5 1245.203 2.0748 15.89 84 23696.11217.673 2.0289 2.98 85 23770.1 1201.463 2.0019 27.17 86 23827.61188.849 1.9809 1.36 87 23887.4 1175.734 1.9590 15.22 88 23919.51168.700 1.9473 15.28 89 24010.2 1148.811 1.9142 0.49 90 24104.21128.207 1.8798 1.77 91 24197.7 1107.716 1.8457 28.22 92 24263.81093.228 1.8216 3.27 93 24321.8 1080.503 1.8004 2.93 94 24373.5 1069.1661.7815 2.83 95 24497.0 1042.094 1.7364 26.10 96 24598.4 1019.871 1.69933.17 97 24626.1 1013.793 1.6892 3.14 98 24676.1 1002.826 1.6709 1.84 9924931.2 946.900 1.5777 2.14 100 24983.0 935.533 1.5588 3.02 101 25226.8882.103 1.4698 0.25 102 25370.4 850.608 1.4173 4.11 103 25412.0 841.4921.4021 4.88 104 25499.7 822.270 1.3701 4.07 105 25556.8 809.746 1.34922.62 106 25639.9 791.527 1.3189 31.95 107 25717.9 774.418 1.2904 2.84108 25790.4 758.539 1.2639 22.85 109 26011.7 710.018 1.1830 4.66 11026070.7 697.082 1.1615 24.95 111 26249.2 657.953 1.0963 31.39 11226536.4 594.981 0.9914 25.26 113 26610.9 578.657 0.9642 0.97 114 26914.0512.196 0.8534 2.04 115 27012.2 490.676 0.8176 25.88 116 27118.1 467.4630.7789 3.69 117 27226.4 443.715 0.7393 1.07 118 28513.4 161.554 0.26920.89 119 28539.8 155.777 0.2596 1.54

TABLE 6.2 HMQC of Y1 peaklist ADDRESS FREQUENCY row [Hz]F1 [PPM]F1 # col[Hz]F2 [PPM]F2 INTENSITY 1 315 16740.193 110.9281 30.72 375 3624.4456.0391 2 352 15855.943 105.0686 20.34 490 2950.420 4.9161 3 45013537.575 89.7061 20.34 490 2950.420 4.9161 4 479 12863.844 85.241612.42 476 3032.063 5.0521 5 479 12863.844 85.2416 17.76 500 2892.3604.8193 6 491 12577.806 83.3462 25.72 487 2969.087 4.9472 7 522 11857.07078.5703 13.55 545 2627.024 4.3772 8 529 11685.553 77.4337 16.93 5042870.087 4.7822 9 535 11537.104 76.4500 15.06 589 2370.150 3.9492 10 55011191.633 74.1608 12.94 421 3355.320 5.5907 11 550 11191.633 74.160815.14 423 3342.807 5.5699 12 569 10733.316 71.1238 24.93 410 3421.2955.7006 13 579 10497.793 69.5631 26.50 526 2740.480 4.5662 14 58610332.951 68.4708 14.53 537 2674.056 4.4556 15 586 10332.951 68.470811.00 596 2330.159 3.8826 16 617 9621.364 63.7555 12.23 643 2051.8863.4189 17 630 9299.908 61.6254 13.25 529 2718.381 4.5294 18 630 9299.90861.6254 14.42 539 2665.365 4.4411 19 669 8391.979 55.6090 13.25 5292718.381 4.5294 20 669 8391.979 55.6090 14.42 539 2665.365 4.4411 21 7207193.972 47.6705 10.83 682 1821.991 3.0358 22 725 7055.427 46.7524 8.84820 1015.280 1.6917 23 770 5994.782 39.7241 21.78 682 1822.241 3.0363 24778 5810.328 38.5019 21.78 682 1822.241 3.0363 25 803 5229.784 34.65499.20 836 918.974 1.5312 26 813 4978.483 32.9897 9.04 866 743.550 1.238927 833 4517.225 29.9332 64.24 847 852.860 1.4211 28 847 4185.850 27.737374.68 861 773.417 1.2887 29 850 4113.891 27.2605 80.21 806 1096.5821.8271 30 850 4113.891 27.2605 31.46 861 773.392 1.2886 31 885 3291.88421.8135 100.00 756 1389.088 2.3145 32 893 3110.641 20.6125 89.09 7961153.093 1.9213 33 893 3110.641 20.6125 50.66 803 1110.425 1.8502 34 8972998.505 19.8694 45.64 803 1111.101 1.8513 35 897 2998.505 19.8694 78.07853 820.195 1.3666 36 919 2497.783 16.5514 72.04 874 699.217 1.1651 37919 2497.783 16.5514 37.18 910 483.491 0.8056 38 926 2336.725 15.484217.70 794 1165.059 1.9412 39 926 2336.725 15.4842 31.53 910 484.3920.8071

TABLE 6.2A 2D NMR (HMQC) data of Y1 Assignment w1 w2 Data Height  1C-H110.904 6.038 24704052  2C-H 105.023 4.914 15964054  3C-H 104.615 5.33911824459  4C-H 89.823 3.243 7951700  5C-H 89.712 3.222 7911944  6C-H85.528 5.038 10494958  7C-H 85.417 5.054 8839478  8C-H 85.338 4.83110297378  9C-H 85.248 4.817 12857784 10C-H 83.397 4.948 19288902 11C-H78.655 4.381 7900396 12C-H 78.602 4.366 8972991 13C-H 77.420 4.78413438428 14C-H 77.007 4.497 7206874 15C-H 76.570 3.951 12216028 16C-H74.994 4.092 11486882 17C-H 74.790 4.106 12904558 18C-H 74.220 5.59310130728 19C-H 74.062 5.574 9532875 20C-H 73.856 6.173 11098625 21C-H73.802 6.156 10393206 22C-H 73.350 4.468 11007188 23C-H 73.277 4.4467281630 24C-H 71.577 4.453 8645994 25C-H 71.219 5.701 24595648 26C-H70.611 3.615 9076031 27C-H 70.067 4.256 9261139 28C-H 69.616 4.56718994736 29C-H 68.771 3.895 8451744 30C-H 68.543 4.455 12573076 31C-H63.679 3.604 8239119 32C-H 63.781 3.415 7458621 33C-H 63.862 3.3937841054 34C-H 62.192 4.312 10795595 35C-H 62.172 4.287 8408334 36C-H62.266 4.162 7944312 37C-H 62.071 4.131 10031945 38C-H 61.811 4.52912476046 39C-H 61.793 4.440 11880108 40C-H 61.705 4.463 8055856 41C-H47.663 3.034 7772454 42C-H 46.740 1.693 8583785 43C-H 39.795 3.03418038864 44C-H 38.453 1.337 7912356 45C-H 34.666 1.567 7317314 46C-H32.884 1.264 7887187 47C-H 29.954 1.421 58253544 48C-H 29.632 1.24216326471 49C-H 29.318 1.117 9248448 50C-H 27.723 1.292 47380240 51C-H27.251 1.831 59260700 52C-H 21.835 2.317 68135008 53C-H 20.604 1.91959543524 54C-H 20.377 1.852 66987324 55C-H 19.850 1.369 68067232 56C-H16.547 1.167 54857956 57C-H 16.223 0.807 25159514 58C-H 15.890 2.02814256620 59C-H 15.674 1.978 12750140 60C-H 15.693 1.940 10562185 61C-H15.378 0.758 16911768 62C-H 15.149 0.799 12756197 63C-H 138.670 5.9858925989 64C-H 137.837 5.881 6871625 65C-H 123.420 5.365 15877691

TABLE 6.3 2D NMR (HMBC) data of Y1 peaklist ADDRESS FREQUENCY row [Hz]F1[PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 148 25871.928 171.4391 11.03362 3702.490 6.1692 2 148 25871.928 171.4391 93.44 756 1390.051 2.3161 3169 25232.139 167.1995 28.04 796 1153.459 1.9219 4 169 25232.139167.1995 29.81 803 1112.088 1.8530 5 293 21555.236 142.8347 25.74 8061097.351 1.8284 6 314 20935.686 138.7293 33.12 786 1215.627 2.0255 7 31420935.686 138.7293 46.88 796 1153.008 1.9212 8 318 20813.068 137.91689.66 786 1213.951 2.0227 9 318 20813.068 137.9168 29.47 792 1175.4381.9585 10 318 20813.068 137.9168 46.19 803 1110.584 1.8505 11 36919321.684 128.0342 13.34 785 1218.100 2.0296 12 369 19321.684 128.034228.29 792 1175.050 1.9579 13 369 19321.684 128.0342 26.99 796 1153.6911.9223 14 369 19321.684 128.0342 65.83 803 1111.953 1.8528 15 48615874.223 105.1898 16.48 477 3026.433 5.0427 16 486 15874.223 105.18989.96 545 2628.184 4.3791 17 486 15874.223 105.1898 10.46 557 2559.5784.2648 18 565 13533.447 89.6787 10.78 491 2942.427 4.9027 19 56513533.447 89.6787 41.26 861 774.469 1.2904 20 565 13533.447 89.678736.06 874 696.834 1.1611 21 587 12903.256 85.5028 20.78 362 3702.8446.1698 22 587 12903.256 85.5028 12.97 375 3625.743 6.0413 23 58712903.256 85.5028 35.15 847 852.970 1.4212 24 587 12903.256 85.502830.07 853 821.094 1.3681 25 622 11874.164 78.6836 9.13 447 3202.8995.3367 26 622 11874.164 78.6836 9.75 563 2519.490 4.1980 27 64711136.594 73.7961 19.44 476 3029.853 5.0484 28 647 11136.594 73.79619.88 625 2159.425 3.5981 29 660 10736.229 71.1431 30.15 873 702.4471.1704 30 668 10508.083 69.6313 11.82 873 702.134 1.1699 31 67310374.060 68.7432 41.29 873 702.539 1.1706 32 709 9310.579 61.6961 9.48588 2374.061 3.9557 33 740 8380.918 55.5357 42.71 861 775.074 1.2914 34740 8380.918 55.5357 30.33 874 696.643 1.1608 35 740 8380.918 55.535721.32 913 469.072 0.7816 36 781 7183.864 47.6035 43.72 847 853.2771.4217 37 781 7183.864 47.6035 35.29 853 821.688 1.3691 38 786 7044.03446.6769 9.09 848 851.746 1.4192 39 786 7044.034 46.6769 8.36 880 661.9831.1030 40 786 7044.034 46.6769 27.00 913 469.396 0.7821 41 813 6236.90141.3285 38.52 806 1097.285 1.8283 42 813 6236.901 41.3285 43.99 910485.026 0.8082 43 822 5962.607 39.5109 32.36 806 1097.624 1.8289 44 8225962.607 39.5109 41.54 861 774.567 1.2906 45 822 5962.607 39.5109 63.83874 696.338 1.1603 46 822 5962.607 39.5109 32.10 910 484.561 0.8074 47828 5792.666 38.3848 9.58 853 821.024 1.3680 48 828 5792.666 38.384839.07 913 469.203 0.7818 49 832 5676.145 37.6127 42.80 847 852.6431.4207 50 832 5676.145 37.6127 53.76 853 821.900 1.3695 51 832 5676.14537.6127 10.57 912 471.157 0.7851 52 837 5531.661 36.6553 8.78 853822.175 1.3699 53 837 5531.661 36.6553 45.56 913 468.520 0.7807 54 8475226.124 34.6307 27.35 805 1099.040 1.8312 55 856 4961.126 32.8747 32.47910 484.632 0.8075 56 871 4515.250 29.9201 7.66 477 3025.946 5.0419 57871 4515.250 29.9201 8.91 837 913.407 1.5219 58 871 4515.250 29.920152.28 853 821.483 1.3688 59 883 4184.067 27.7255 8.46 795 1160.8191.9342 60 883 4184.067 27.7255 11.68 850 836.992 1.3946 61 883 4184.06727.7255 77.80 874 696.591 1.1607 62 913 3294.604 21.8315 16.90 7451453.362 2.4216 63 913 3294.604 21.8315 15.41 767 1324.146 2.2063 64 9232998.099 19.8668 12.57 477 3025.408 5.0410 65 923 2998.099 19.8668 9.99683 1819.240 3.0313 66 923 2998.099 19.8668 18.02 842 883.190 1.4716 67923 2998.099 19.8668 51.49 847 853.156 1.4215 68 923 2998.099 19.866816.19 864 757.915 1.2629 69 923 2998.099 19.8668 10.30 879 666.3281.1103 70 940 2480.249 16.4352 42.66 861 775.278 1.2918 71 940 2480.24916.4352 14.60 884 636.584 1.0607 72 940 2480.249 16.4352 12.91 900544.736 0.9077 73 940 2480.249 16.4352 12.28 921 420.806 0.7012 74 9462313.568 15.3307 14.80 902 531.201 0.8851 75 946 2313.568 15.3307 12.79923 407.732 0.6794

TABLE 6.4 2D NMR (COSY) data of Y1. Assignment w1 w2 Data Height  1H-H6.704 6.322 9224233  2H-H 6.678 6.372 9898637  3H-H 6.419 4.456 20015776 4H-H 6.368 6.681 11333652  5H-H 6.320 6.706 11307411  6H-H 6.171 5.044134583264  7H-H 6.043 4.952 12934876  8H-H 5.980 2.030 80531096  9H-H5.980 2.021 105609248 10H-H 5.881 1.963 81350000 11H-H 5.881 1.95275881512 12H-H 5.701 5.700 215204304 13H-H 5.700 5.590 18340750 14H-H5.699 5.575 15251625 15H-H 5.582 5.701 24882492 16H-H 5.583 4.93032200604 17H-H 5.583 4.269 54166860 18H-H 5.582 4.260 100437000 19H-H5.582 4.251 50904084 20H-H 5.335 4.478 54908116 21H-H 5.335 4.46248000284 22H-H 5.334 4.100 20768318 23H-H 5.048 6.174 89161792 24H-H4.949 4.792 11766272 25H-H 4.950 4.782 9033592 26H-H 4.930 5.58317147768 27H-H 4.929 4.269 30517720 28H-H 4.927 4.251 28975240 29H-H4.901 4.390 27806448 30H-H 4.900 4.373 34077108 31H-H 4.831 4.79099849200 32H-H 4.820 4.310 7275091 33H-H 4.819 4.296 7797132 34H-H 4.8224.157 13857122 35H-H 4.821 4.138 18211468 36H-H 4.794 4.953 3063057837H-H 4.787 4.827 86722272 38H-H 4.568 4.109 17535982 39H-H 4.567 4.09315653847 40H-H 4.542 4.466 32239438 41H-H 4.522 4.453 145233152 42H-H4.517 4.205 7769614 43H-H 4.523 3.952 73572400 44H-H 4.507 4.43611835048 45H-H 4.468 5.337 79724560 46H-H 4.468 4.099 83781344 47H-H4.461 3.954 44029948 48H-H 4.454 6.528 9453154 49H-H 4.454 6.41879266688 50H-H 4.450 4.381 13566321 51H-H 4.449 4.321 8581714 52H-H4.444 4.214 29697092 53H-H 4.445 4.205 23505830 54H-H 4.443 4.19523169768 55H-H 4.382 4.902 75968808 56H-H 4.380 4.214 34194940 57H-H4.379 4.204 34462264 58H-H 4.380 4.195 34413552 59H-H 4.302 4.82316076232 60H-H 4.302 4.148 221634448 61H-H 4.262 5.582 80620088 62H-H4.261 4.930 59383108 63H-H 4.203 4.900 12911051 64H-H 4.204 4.43320195584 65H-H 4.150 4.832 17371252 66H-H 4.153 4.819 18323128 67H-H4.149 4.305 216562864 68H-H 4.099 5.337 25030224 69H-H 4.100 4.57136121208 70H-H 4.101 4.472 68659520 71H-H 4.095 4.460 36864516 72H-H4.028 4.468 7964866 73H-H 4.028 4.458 8388422 74H-H 3.961 4.463 4189777675H-H 3.953 4.539 34935532 76H-H 3.952 4.523 50083884 77H-H 3.953 4.44724637258 78H-H 3.893 1.162 59572844 79H-H 3.887 1.175 95668312 80H-H3.766 3.516 11119055 81H-H 3.744 3.494 6884830 82H-H 3.598 3.413146852352 83H-H 3.598 3.404 128194976 84H-H 3.517 3.767 10865892 85H-H3.508 3.759 9535602 86H-H 3.480 3.741 8054603 87H-H 3.475 3.734 626886388H-H 3.409 3.599 107289744 89H-H 3.406 3.592 110786000 90H-H 3.2292.109 12003553 91H-H 2.110 1.838 8593652 92H-H 2.107 1.822 1147412893H-H 2.107 1.811 10279236 94H-H 2.105 1.794 8116300 95H-H 2.029 5.99247626580 96H-H 2.027 5.984 47768532 97H-H 2.026 5.972 51874608 98H-H2.030 2.118 7536440 99H-H 2.027 1.919 89540200 100H-H  2.024 5.96441580316 101H-H  1.959 5.891 49355596 102H-H  1.955 5.882 46234528103H-H  1.956 5.870 51174608 104H-H  1.954 1.848 77092128 105H-H  1.9232.030 54674196 106H-H  1.917 2.020 54160620 107H-H  1.848 1.958 62600468108H-H  1.834 1.756 16637570 109H-H  1.829 1.732 26336268 110H-H  1.8281.711 15651960 111H-H  1.835 1.677 25025304 112H-H  1.830 1.553106561752 113H-H  1.828 1.536 92597192 114H-H  1.820 3.231 15325426115H-H  1.819 3.220 13169861 116H-H  1.818 2.114 14925469 117H-H  1.8182.106 16020545 118H-H  1.742 1.848 24286072 119H-H  1.745 1.827 35995120120H-H  1.669 1.827 14851102 121H-H  1.546 1.834 96039856 122H-H  1.5431.819 72780528 123H-H  1.545 1.270 7218780 124H-H  1.546 1.255 36723868125H-H  1.544 1.241 22604678 126H-H  1.476 1.271 6846019 127H-H  1.4771.255 10952480 128H-H  1.471 1.242 8182750 129H-H  1.474 0.727 7531610130H-H  1.348 0.812 23199942 131H-H  1.348 0.796 27081584 132H-H  1.3440.784 22663138 133H-H  1.260 1.540 12084268 134H-H  1.263 1.489 14416738135H-H  1.263 1.474 17048864 136H-H  1.267 0.736 16594936 137H-H  1.2660.719 16949332 138H-H  1.252 1.558 13171360 139H-H  1.250 1.525 19080828140H-H  1.237 0.837 11506187 141H-H  1.167 3.901 86146744 142H-H  1.1673.877 80709560 143H-H  0.840 1.234 8462904 144H-H  0.798 1.351 39178912145H-H  0.798 1.342 32789168 146H-H  0.730 1.482 11378742 147H-H  0.7311.270 11791943 148H-H  0.729 1.255 15935769 149H-H  4.460 4.531 92968608150H-H  4.203 4.382 45014720 151H-H  4.203 4.373 32816148 152H-H  1.7951.675 4628933

TABLE 6.5 Proton NMR chemical shift data of Y1 FREQUENCY # ADDRESS [Hz][PPM] INTENSITY 1 5803.6 5312.007 8.8510 1.05 2 6201.8 5224.701 8.7055180.00 3 6517.2 5155.558 8.5903 0.09 4 6612.7 5134.626 8.5554 1.05 58992.9 4612.799 7.6859 0.41 6 9356.8 4533.029 7.5530 62.60 7 9733.14450.514 7.4155 0.40 8 9952.7 4402.383 7.3353 0.13 9 9996.2 4392.8327.3194 0.90 10 10361.5 4312.746 7.1860 134.51 11 10641.7 4251.327 7.08370.05 12 10739.1 4229.980 7.0481 0.87 13 12483.6 3847.521 6.4108 0.10 1412504.0 3843.034 6.4033 0.10 15 13117.1 3708.630 6.1794 0.31 16 13161.73698.843 6.1631 0.31 17 13503.5 3623.905 6.0382 0.38 18 13617.2 3598.9885.9967 0.08 19 13649.9 3591.811 5.9848 0.19 20 13679.2 3585.404 5.97410.18 21 13712.9 3577.998 5.9617 0.08 22 13886.8 3539.884 5.8982 0.09 2313920.0 3532.594 5.8861 0.19 24 13952.5 3525.479 5.8742 0.17 25 13983.63518.658 5.8629 0.06 26 14413.7 3424.364 5.7058 0.36 27 14428.7 3421.0805.7003 0.36 28 14720.0 3357.206 5.5939 0.19 29 14735.6 3353.801 5.58820.18 30 14766.4 3347.042 5.5769 0.20 31 14781.9 3343.652 5.5713 0.17 3215028.7 3289.533 5.4811 0.05 33 15343.4 3220.543 5.3661 0.33 34 15404.63207.131 5.3438 0.25 35 15439.6 3199.461 5.3310 0.25 36 16193.8 3034.1115.0555 0.14 37 16238.8 3024.240 5.0391 0.15 38 16381.2 2993.014 4.98700.17 39 16518.3 2962.967 4.9370 0.12 40 16553.7 2955.209 4.9240 0.12 4116599.3 2945.198 4.9074 0.08 42 16633.5 2937.700 4.8949 0.08 43 16843.22891.736 4.8183 0.18 44 16905.3 2878.117 4.7956 0.17 45 16927.0 2873.3704.7877 0.19 46 17523.6 2742.566 4.5697 0.33 47 17586.3 2728.818 4.54680.18 48 17617.6 2721.965 4.5354 0.23 49 17635.4 2718.066 4.5289 0.31 5017665.9 2711.371 4.5177 0.32 51 17716.2 2700.349 4.4994 0.21 52 17760.52690.632 4.4832 0.20 53 17810.9 2679.588 4.4648 0.55 54 17835.7 2674.1414.4557 0.72 55 17857.9 2669.267 4.4476 0.54 56 18007.8 2636.409 4.39280.17 57 18046.2 2627.986 4.3788 0.26 58 18083.2 2619.886 4.3653 0.19 5918189.9 2596.493 4.3263 0.11 60 18233.6 2586.912 4.3104 0.27 61 18277.82577.209 4.2942 0.31 62 18331.5 2565.443 4.2746 0.21 63 18367.6 2557.5424.2614 0.23 64 18412.4 2547.721 4.2451 0.19 65 18458.8 2537.540 4.22810.10 66 18491.9 2530.283 4.2160 0.21 67 18532.6 2521.359 4.2011 0.28 6818567.2 2513.770 4.1885 0.14 69 18645.4 2496.625 4.1599 0.25 70 18666.52491.997 4.1522 0.25 71 18699.9 2484.686 4.1400 0.24 72 18721.5 2479.9434.1321 0.19 73 18778.7 2467.413 4.1113 0.20 74 18792.8 2464.305 4.10610.20 75 18821.9 2457.939 4.0955 0.19 76 18835.7 2454.909 4.0904 0.18 7719186.2 2378.063 3.9624 0.17 78 19213.7 2372.029 3.9523 0.29 79 19239.62366.350 3.9429 0.16 80 19338.7 2344.635 3.9067 0.08 81 19366.7 2338.4903.8964 0.22 82 19396.9 2331.872 3.8854 0.21 83 19428.2 2325.007 3.87400.06 84 20015.2 2196.324 3.6596 0.05 85 20048.6 2188.994 3.6474 0.08 8620131.1 2170.900 3.6172 0.13 87 20161.7 2164.211 3.6061 0.28 88 20208.12154.040 3.5891 0.31 89 20679.1 2050.765 3.4170 0.24 90 20727.6 2040.1353.3993 0.23 91 20782.4 2028.119 3.3793 0.08 92 21179.2 1941.123 3.23430.14 93 21213.6 1933.579 3.2218 0.13 94 21586.4 1851.862 3.0856 0.07 9521649.4 1838.049 3.0626 0.09 96 21715.8 1823.490 3.0383 0.67 97 21747.51816.527 3.0267 0.23 98 23153.9 1508.199 2.5130 0.06 99 23693.5 1389.9162.3159 2.02 100 24231.4 1271.971 2.1194 0.22 101 24282.2 1260.831 2.10080.22 102 24341.3 1247.878 2.0792 0.16 103 24413.6 1232.024 2.0528 0.15104 24464.0 1220.989 2.0344 0.83 105 24468.7 1219.963 2.0327 0.83 10624496.7 1213.827 2.0225 0.82 107 24540.5 1204.212 2.0065 0.24 10824614.2 1188.066 1.9796 0.24 109 24658.3 1178.391 1.9635 0.88 11024686.0 1172.311 1.9533 0.86 111 24690.9 1171.249 1.9516 0.85 11224774.5 1152.908 1.9210 1.65 113 24824.7 1141.905 1.9027 0.22 11424966.5 1110.815 1.8509 2.07 115 25021.9 1098.679 1.8306 2.09 11625154.1 1069.689 1.7823 0.22 117 25213.5 1056.676 1.7607 0.20 11825280.4 1042.010 1.7362 0.33 119 25347.2 1027.363 1.7118 0.24 12025396.2 1016.604 1.6939 0.38 121 25591.3 973.839 1.6226 0.16 122 25626.4966.149 1.6098 0.22 123 25673.7 955.785 1.5926 0.16 124 25759.1 937.0611.5614 0.45 125 25837.0 919.968 1.5329 0.32 126 25932.6 899.023 1.49800.22 127 25995.9 885.130 1.4748 0.28 128 26142.6 852.967 1.4212 1.52 12926284.2 821.936 1.3695 1.99 130 26355.3 806.354 1.3436 0.49 131 26405.2795.414 1.3253 0.53 132 26437.0 788.423 1.3137 0.61 133 26495.4 775.6201.2924 1.83 134 26547.6 764.186 1.2733 0.62 135 26578.6 757.400 1.26200.75 136 26635.0 745.018 1.2414 0.66 137 26710.2 728.529 1.2139 0.35 13826742.2 721.515 1.2022 0.34 139 26774.1 714.532 1.1906 0.38 140 26818.0704.909 1.1745 1.47 141 26852.6 697.329 1.1619 2.39 142 26939.0 678.3821.1303 0.28 143 26967.1 672.226 1.1201 0.28 144 26987.0 667.851 1.11280.31 145 27035.8 657.144 1.0949 0.25 146 27059.9 651.863 1.0861 0.21 14727091.3 644.992 1.0747 0.18 148 27116.3 639.514 1.0656 0.13 149 27306.1597.891 0.9962 0.21 150 27331.8 592.271 0.9869 0.20 151 27362.1 585.6220.9758 0.18 152 27426.7 571.457 0.9522 0.07 153 27465.2 563.025 0.93810.12 154 27550.1 544.395 0.9071 0.06 155 27604.4 532.507 0.8873 0.09 15627723.7 506.347 0.8437 0.23 157 27778.4 494.349 0.8237 0.48 158 27825.7483.975 0.8064 1.88 159 27892.5 469.335 0.7820 1.79 160 27999.1 445.9680.7431 0.28 161 28054.1 433.917 0.7230 0.23 162 28134.1 416.375 0.69380.08 163 28228.5 395.678 0.6593 0.07 164 28262.5 388.209 0.6468 0.09

TABLE 8.2 2D NMR (HMQC) of R1 peaklist. ADDRESS FREQUENCY row [Hz]F1[PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 151 20624.836 136.6695 4.88 3973494.332 5.8223 2 157 20461.906 135.5898 67.68 220 4534.469 7.5554 3 23418646.779 123.5620 100.00 258 4313.193 7.1867 4 234 18646.779 123.56209.18 445 3213.863 5.3550 5 343 16069.902 106.4864 7.58 496 2913.3674.8543 6 343 16069.902 106.4864 7.61 498 2904.202 4.8390 7 350 15895.188105.3287 7.99 471 3059.891 5.0985 8 350 15895.188 105.3287 9.11 4733050.642 5.0830 9 350 15895.188 105.3287 8.79 481 3004.427 5.0060 10 36315605.384 103.4083 7.96 510 2835.541 4.7246 11 380 15192.972 100.675516.30 326 3912.206 6.5186 12 454 13447.552 89.1095 4.35 656 1974.6103.2901 13 454 13447.552 89.1095 4.45 659 1958.017 3.2625 14 51512025.116 79.6838 4.85 561 2533.049 4.2206 15 518 11934.442 79.0830 4.52389 3540.938 5.9000 16 524 11802.268 78.2071 12.80 562 2525.183 4.207517 524 11802.268 78.2071 11.92 594 2340.880 3.9004 18 534 11563.50176.6250 4.43 571 2474.883 4.1237 19 534 11563.501 76.6250 3.52 5882372.543 3.9532 20 534 11563.501 76.6250 5.94 610 2246.165 3.7426 21 53411563.501 76.6250 6.60 612 2234.362 3.7229 22 540 11437.312 75.788820.96 574 2457.347 4.0945 23 545 11309.472 74.9417 5.29 553 2580.6284.2999 24 545 11309.472 74.9417 6.15 555 2569.264 4.2810 25 54511309.472 74.9417 3.23 561 2533.966 4.2222 26 545 11309.472 74.9417 9.38582 2411.457 4.0180 27 551 11175.793 74.0558 3.74 550 2597.570 4.3281 28551 11175.793 74.0558 7.04 552 2585.925 4.3087 29 551 11175.793 74.05583.84 554 2574.515 4.2897 30 556 11047.378 73.2049 5.90 586 2386.1663.9759 31 562 10907.159 72.2758 15.78 511 2826.203 4.7091 32 56210907.159 72.2758 5.35 520 2777.020 4.6271 33 567 10784.600 71.463612.61 562 2525.038 4.2073 34 567 10784.600 71.4636 4.33 567 2497.6644.1617 35 577 10543.526 69.8662 4.00 497 2908.530 4.8463 36 57710543.526 69.8662 3.60 500 2892.560 4.8196 37 577 10543.526 69.8662 5.98514 2810.206 4.6824 38 577 10543.526 69.8662 5.96 516 2796.413 4.6594 39577 10543.526 69.8662 6.72 548 2609.404 4.3478 40 624 9434.661 62.518311.62 532 2705.029 4.5072 41 624 9434.661 62.5183 8.01 534 2689.7274.4817 42 624 9434.661 62.5183 7.54 547 2616.577 4.3598 43 669 8381.18055.5375 4.80 919 431.689 0.7193 44 669 8381.180 55.5375 5.21 922 414.8810.6913 45 719 7203.831 47.7358 3.32 827 972.057 1.6197 46 728 6986.54246.2960 3.85 777 1265.417 2.1085 47 728 6986.542 46.2960 3.19 859784.626 1.3074 48 728 6986.542 46.2960 3.45 862 764.970 1.2746 49 7596273.788 41.5729 3.64 724 1575.188 2.6246 50 759 6273.788 41.5729 3.54728 1556.033 2.5927 51 817 4904.791 32.5014 9.21 849 844.868 1.4077 52831 4556.851 30.1957 40.19 868 732.276 1.2201 53 846 4212.870 27.916445.73 870 719.170 1.1983 54 858 3932.408 26.0579 45.69 865 749.6751.2491 55 893 3102.798 20.5605 37.60 808 1085.904 1.8094 56 900 2935.04419.4489 42.87 864 755.857 1.2594 57 905 2821.322 18.6953 25.73 8081086.381 1.8102 58 918 2523.121 16.7193 36.59 883 644.770 1.0743 59 9182523.121 16.7193 43.39 895 571.319 0.9519 60 924 2368.333 15.6936 21.68794 1163.335 1.9384 61 924 2368.333 15.6936 45.90 895 573.050 0.9548

TABLE 8.3 HMBC R1 peaklist DU = C:/Bruker/XWIN-NMR, USER = guest, NAME =chan, EXPNO = 26, PROCNO = 1 F1PLO = 173.650 ppm, F1PHI = 11.502 ppm,F2PLO = 6.832 ppm, F2PHI = 0.515 ppm MI = 5.00 cm, MAXI = 10000.00 cm,PC = 3.000 ADDRESS FREQUENCY row [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2INTENSITY 1 166 25330.719 167.8561 389 3542.372 5.9024 11.00 2 16625330.719 167.8561 808 1085.572 1.8088 37.94 3 293 21569.176 142.9299865 750.004 1.2497 39.69 4 325 20624.461 136.6697 795 1160.826 1.934254.15 5 325 20624.461 136.6697 808 1085.191 1.8082 67.95 6 366 19424.748128.7197 795 1161.341 1.9351 54.29 7 366 19424.748 128.7197 808 1085.2931.8083 100.00 8 479 16074.168 106.5168 587 2382.396 3.9696 17.03 9 48515911.199 105.4369 582 2411.570 4.0182 13.64 10 495 15610.658 103.4453559 2544.112 4.2391 7.40 11 495 15610.658 103.4453 574 2457.717 4.09517.72 12 509 15196.733 100.7024 311 3999.566 6.6642 10.46 13 50915196.733 100.7024 341 3825.973 6.3749 10.97 14 568 13447.739 89.1126497 2908.018 4.8454 13.78 15 568 13447.739 89.1126 870 718.459 1.197152.71 16 568 13447.739 89.1126 896 570.271 0.9502 33.62 17 616 12037.52179.7676 560 2540.187 4.2325 10.39 18 616 12037.521 79.7676 567 2496.4234.1596 12.77 19 620 11934.375 79.0841 557 2555.670 4.2583 16.69 20 62011934.375 79.0841 586 2384.236 3.9727 18.17 21 624 11794.329 78.1561 5612532.465 4.2196 29.22 22 624 11794.329 78.1561 581 2415.675 4.0251 13.9023 632 11561.078 76.6104 554 2575.363 4.2911 11.27 24 632 11561.07876.6104 861 774.214 1.2900 8.95 25 632 11561.078 76.6104 864 757.6311.2624 31.05 26 632 11561.078 76.6104 868 733.017 1.2214 52.22 27 64011319.147 75.0073 562 2529.498 4.2147 14.09 28 640 11319.147 75.0073 5742457.979 4.0955 12.03 29 640 11319.147 75.0073 611 2238.440 3.7297 7.3530 645 11173.489 74.0421 511 2826.331 4.7093 5.08 31 645 11173.48974.0421 807 1087.742 1.8124 27.89 32 650 11046.945 73.2035 389 3541.0205.9001 14.57 33 655 10904.058 72.2566 326 3913.536 6.5208 23.27 34 65510904.058 72.2566 552 2586.768 4.3101 9.96 35 655 10904.058 72.2566 5632521.903 4.2021 6.06 36 659 10780.204 71.4359 532 2702.900 4.5036 6.9337 659 10780.204 71.4359 534 2692.127 4.4857 8.19 38 659 10780.20471.4359 563 2520.533 4.1998 48.21 39 667 10532.287 69.7931 472 3054.4955.0895 11.46 40 667 10532.287 69.7931 480 3007.639 5.0114 18.84 41 67110425.301 69.0841 326 3913.495 6.5208 32.61 42 671 10425.301 69.0841 5522586.054 4.3089 7.93 43 671 10425.301 69.0841 807 1088.313 1.8134 58.1544 705 9430.492 62.4919 560 2539.835 4.2319 5.37 45 740 8372.175 55.4789870 718.660 1.1974 38.08 46 740 8372.175 55.4789 895 571.407 0.952144.50 47 780 7199.307 47.7068 883 644.628 1.0741 27.47 48 780 7199.30747.7068 895 572.447 0.9538 31.41 49 788 6981.523 46.2636 864 757.7671.2626 51.63 50 788 6981.523 46.2636 868 732.960 1.2213 78.63 51 8046495.941 43.0459 574 2458.027 4.0956 8.82 52 804 6495.941 43.0459 861774.879 1.2911 8.89 53 804 6495.941 43.0459 867 737.966 1.2296 6.55 54810 6313.906 41.8396 445 3213.454 5.3543 8.33 55 810 6313.906 41.8396755 1397.464 2.3285 5.40 56 810 6313.906 41.8396 865 749.894 1.249542.24 57 810 6313.906 41.8396 883 644.620 1.0741 52.86 58 814 6189.25641.0136 865 749.287 1.2485 6.31 59 814 6189.256 41.0136 883 643.3621.0720 8.62 60 817 6120.849 40.5603 865 749.159 1.2483 15.69 61 8176120.849 40.5603 883 644.286 1.0735 17.94 62 817 6120.849 40.5603 896570.189 0.9501 6.27 63 819 6053.888 40.1166 865 749.605 1.2490 43.77 64819 6053.888 40.1166 883 644.397 1.0737 47.98 65 819 6053.888 40.1166896 570.067 0.9499 8.91 66 824 5921.430 39.2388 864 754.133 1.2566 5.8067 824 5921.430 39.2388 870 718.264 1.1968 70.97 68 824 5921.430 39.2388896 569.449 0.9488 65.30 69 824 5921.430 39.2388 920 427.451 0.7122 6.7070 824 5921.430 39.2388 922 414.558 0.6907 7.41 71 827 5818.316 38.5556870 718.161 1.1966 8.71 72 827 5818.316 38.5556 895 572.960 0.9547 35.7873 829 5749.308 38.0983 896 570.060 0.9498 8.61 74 837 5529.916 36.6444829 959.437 1.5986 5.22 75 837 5529.916 36.6444 864 755.929 1.2595 12.1576 837 5529.916 36.6444 868 732.618 1.2207 8.77 77 837 5529.916 36.6444895 572.369 0.9537 51.81 78 837 5529.916 36.6444 920 427.331 0.7120 5.3679 837 5529.916 36.6444 922 414.571 0.6908 6.76 80 840 5443.605 36.0725777 1264.891 2.1076 5.25 81 840 5443.605 36.0725 864 757.333 1.261968.88 82 840 5443.605 36.0725 868 732.776 1.2210 83.66 83 840 5443.60536.0725 895 571.988 0.9531 11.13 84 858 4903.620 32.4942 883 644.4231.0738 38.16 85 870 4556.862 30.1964 611 2240.976 3.7340 8.07 86 8704556.862 30.1964 857 794.987 1.3246 18.78 87 870 4556.862 30.1964 864756.793 1.2610 79.89 88 870 4556.862 30.1964 878 670.310 1.1169 18.43 89872 4487.079 29.7340 864 756.125 1.2599 16.38 90 874 4422.445 29.3057863 758.384 1.2636 8.16 91 877 4356.193 28.8667 864 755.084 1.2581 8.7992 877 4356.193 28.8667 896 569.898 0.9496 6.22 93 882 4211.267 27.9063860 779.212 1.2983 19.55 94 882 4211.267 27.9063 864 754.574 1.2573 5.5995 882 4211.267 27.9063 881 654.994 1.0914 19.01 96 882 4211.267 27.9063896 570.162 0.9500 87.84 97 891 3938.165 26.0966 854 812.119 1.353219.50 98 891 3938.165 26.0966 865 751.828 1.2527 10.44 99 891 3938.16526.0966 876 686.003 1.1430 17.89 100 891 3938.165 26.0966 896 569.8520.9495 5.20 101 893 3876.630 25.6888 865 749.874 1.2495 40.70 102 8933876.630 25.6888 876 686.400 1.1437 5.01 103 919 3095.315 20.5114 3983488.142 5.8120 7.58 104 919 3095.315 20.5114 797 1150.511 1.9170 9.43105 919 3095.315 20.5114 818 1022.904 1.7044 9.18 106 919 3095.31520.5114 868 732.855 1.2211 5.53 107 925 2937.158 19.4633 611 2240.9103.7339 13.27 108 925 2937.158 19.4633 777 1265.381 2.1084 11.35 109 9252937.158 19.4633 780 1248.250 2.0799 5.80 110 925 2937.158 19.4633 853818.856 1.3644 23.23 111 925 2937.158 19.4633 868 733.355 1.2219 89.32112 925 2937.158 19.4633 875 693.473 1.1555 20.36 113 932 2710.75617.9630 554 2574.880 4.2903 6.54 114 932 2710.756 17.9630 849 843.9161.4062 5.24 115 932 2710.756 17.9630 868 732.674 1.2208 6.51 116 9392527.765 16.7504 829 960.874 1.6010 6.70 117 939 2527.765 16.7504 870718.686 1.1975 83.68 118 939 2527.765 16.7504 885 631.287 1.0519 21.97119 939 2527.765 16.7504 893 583.038 0.9715 14.30 120 939 2527.76516.7504 906 506.673 0.8442 22.28 121 939 2527.765 16.7504 920 426.0440.7099 7.71 122 939 2527.765 16.7504 922 414.883 0.6913 10.74 123 9442376.340 15.7470 784 1224.570 2.0404 9.28 124 944 2376.340 15.7470 8061096.659 1.8273 8.54 125 944 2376.340 15.7470 870 718.378 1.1970 6.60126 944 2376.340 15.7470 885 633.381 1.0554 19.99 127 944 2376.34015.7470 906 509.452 0.8489 20.05 128 944 2376.340 15.7470 920 426.9880.7115 8.96 129 944 2376.340 15.7470 922 415.107 0.6917 10.34

TABLE 8.4 2D NMR (cosy) of R1 peak list Assignment w1 w2 Data Height 16.521 6.522 257252096 2 6.522 4.711 7840252 3 6.129 0.959 3905950 45.898 5.901 32367934 5 5.902 4.847 10836326 6 5.902 4.258 10138593 75.899 4.127 9360363 8 5.906 3.975 16282743 9 5.821 1.810 7215536 105.817 5.822 31028110 11 5.817 1.944 43526536 12 5.816 1.929 44815556 135.355 5.355 178856592 14 5.094 5.102 109124320 15 5.094 5.085 11664912816 5.092 4.205 15712139 17 5.093 4.048 38195456 18 5.011 5.021 11261235219 5.010 5.004 121053544 20 5.008 4.210 14216868 21 5.011 4.021 3815558422 4.847 5.913 5875469 23 4.845 5.900 4787344 24 4.845 5.889 7478266 254.841 4.855 118994368 26 4.836 4.823 47239140 27 4.836 4.369 92117104 284.831 4.134 9652155 29 4.834 4.116 7044446 30 4.844 3.987 44245188 314.845 3.962 44209660 32 4.798 4.795 12493983 33 4.795 4.621 15346245 344.796 1.821 57265952 35 4.796 1.809 75309312 36 4.792 4.312 21750568 374.753 4.360 24472454 38 4.725 4.733 72665064 39 4.721 4.719 99191696 404.727 4.226 50380324 41 4.709 6.523 22903380 42 4.702 4.629 20594276 434.707 4.612 19038358 44 4.706 4.326 11671381 45 4.670 4.688 34985344 464.669 4.660 40038520 47 4.669 4.355 94428136 48 4.670 3.958 8587019 494.672 3.941 7812038 50 4.620 4.800 8615562 51 4.619 4.711 21326744 524.618 4.633 29866052 53 4.619 4.607 44064012 54 4.621 4.312 23669784 554.584 4.355 27276566 56 4.566 4.365 23402122 57 4.492 4.513 55339136 584.491 4.482 71439344 59 4.496 4.370 164934224 60 4.495 4.360 17406252861 4.499 3.912 17609992 62 4.498 3.896 17924064 63 4.377 4.349 2472012864 4.365 4.843 97382152 65 4.364 4.829 127752656 66 4.356 4.680 6786768067 4.355 4.667 81378376 68 4.361 4.491 211941920 69 4.374 4.393 3844238470 4.364 4.372 27706436 71 4.358 4.335 31327086 72 4.364 4.137 1487634573 4.363 4.113 13081764 74 4.355 3.960 10728486 75 4.357 3.940 955240876 4.371 3.913 20295026 77 4.372 3.895 17021364 78 4.309 4.799 2590551879 4.309 4.619 43016120 80 4.306 4.310 56206892 81 4.294 3.742 8058192882 4.295 3.731 101483432 83 4.280 4.354 9482896 84 4.251 5.915 1737460685 4.250 5.901 10701927 86 4.251 5.887 17140758 87 4.253 4.259 3244448088 4.258 4.125 31047850 89 4.257 3.976 5638105 90 4.229 4.730 3464248491 4.207 5.092 13039638 92 4.209 5.012 11226875 93 4.217 4.179 3354465494 4.209 4.058 28007252 95 4.207 4.047 46281960 96 4.211 4.020 3387626097 4.207 4.005 21146824 98 4.210 3.904 80923600 99 4.204 4.365 9066176100 4.202 4.032 40055832 101 4.167 3.951 18178542 102 4.150 4.20911484229 103 4.126 5.912 6719130 104 4.122 5.890 7671593 105 4.126 4.3789045784 106 4.127 4.273 34269312 107 4.119 4.258 32734018 108 4.1334.246 34046856 109 4.131 4.140 18941800 110 4.116 4.355 11770206 1114.092 4.096 279326848 112 4.043 5.095 84940104 113 4.035 4.208 50268828114 4.042 4.183 15655260 115 4.043 4.064 26875002 116 4.019 5.01577663464 117 4.015 4.241 9036684 118 4.027 4.226 11811450 119 4.0164.037 19835598 120 3.973 5.915 24059896 121 3.974 5.901 20609464 1223.973 5.887 22835978 123 3.975 4.853 37923332 124 3.977 3.995 18827896125 3.949 4.682 5809583 126 3.950 4.371 10668304 127 3.953 4.34815916606 128 3.949 4.175 18189762 129 3.948 4.161 30027716 130 3.9534.149 19945408 131 3.940 3.966 14571032 132 3.903 4.505 10048077 1333.903 4.488 14760281 134 3.903 4.387 19898852 135 3.912 4.342 16459215136 3.904 4.239 12064081 137 3.907 4.214 108106008 138 3.898 4.36432756190 139 3.900 4.199 27604272 140 3.901 3.917 43085776 141 3.8973.888 38415896 142 3.735 4.300 93317560 143 3.736 4.290 98467152 1443.736 3.748 65327612 145 3.735 3.725 50388764 146 3.270 3.283 17369858147 3.269 3.267 22139430 148 3.271 2.354 5853844 149 3.271 2.342 7970904150 3.271 1.912 5107326 151 3.271 1.896 5093256 152 2.602 2.631 5486520153 2.600 2.598 5094291 154 2.601 2.590 7143968 155 2.602 2.132 14357642156 2.602 2.109 26185532 157 2.601 2.086 14739034 158 2.603 1.28914662965 159 2.341 3.287 5980422 160 2.342 3.264 5054491 161 2.334 2.3605303651 162 2.336 1.932 6500135 163 2.334 1.872 7760766 164 2.325 2.34113008529 165 2.325 2.315 11101831 166 2.328 1.923 6345431 167 2.3261.895 9356538 168 2.289 2.294 14262412 169 2.186 2.193 4223454 170 2.1032.620 30164346 171 2.103 2.598 27444406 172 2.101 2.109 7249900 1732.105 1.307 22762886 174 2.102 1.300 18897670 175 2.103 1.279 18250726176 2.070 2.078 4104108 177 1.967 1.969 13308390 178 1.977 1.609 7894784179 1.956 1.909 11551598 180 1.961 1.874 10608534 181 1.965 1.6255946444 182 1.931 5.834 39974876 183 1.931 5.824 42157648 184 1.9325.812 40474008 185 1.931 5.798 30554264 186 1.931 2.320 12384753 1871.931 1.942 228192416 188 1.931 1.810 59524928 189 1.913 2.339 13306613190 1.902 2.001 6772636 191 1.895 3.287 9744711 192 1.896 3.264 9357915193 1.897 2.362 8772959 194 1.886 1.973 16266749 195 1.885 1.885 5269322196 1.882 1.616 15011650 197 1.883 1.242 8077304 198 1.878 1.219 8767326199 1.818 5.840 3029832 200 1.805 5.829 3553817 201 1.804 5.808 3418502202 1.805 4.822 22320414 203 1.805 4.813 19315348 204 1.806 4.79727156710 205 1.806 4.785 23402710 206 1.806 4.774 22620674 207 1.8064.322 15274810 208 1.806 4.312 11189881 209 1.806 4.302 17586586 2101.802 1.943 42426312 211 1.804 1.932 30620986 212 1.804 1.810 1349507072213 1.776 1.778 8674281 214 1.703 1.710 5954890 215 1.696 1.702 9409535216 1.613 1.911 8734371 217 1.611 1.635 17314310 218 1.609 1.61029144662 219 1.609 1.599 16356248 220 1.613 1.032 9463035 221 1.6141.017 7358422 222 1.613 0.991 9572058 223 1.406 1.303 9782705 224 1.3951.406 70684776 225 1.393 1.385 22170084 226 1.390 1.293 11202919 2271.307 1.318 7420988 228 1.285 2.609 28951250 229 1.284 2.133 16431712230 1.285 2.110 23106420 231 1.283 2.083 14481833 232 1.279 1.40711364364 233 1.288 1.384 6974150 234 1.286 1.299 18845462 235 1.2880.712 21876642 236 1.288 0.696 27076318 237 1.251 1.260 768046912 2381.221 1.927 5903658 239 1.217 1.223 1002405376 240 1.188 1.197 664805376241 1.168 1.171 16019256 242 1.137 1.145 30540644 243 1.113 1.12037708240 244 1.070 1.077 775147520 245 1.058 1.058 17850510 246 1.0042.349 5175155 247 1.003 1.916 6850918 248 1.003 1.892 5963474 249 1.0051.630 21215516 250 1.005 1.610 23547650 251 1.000 1.008 25525576 2521.016 0.959 33680632 253 0.985 0.999 11598959 254 0.963 0.985 13491636255 0.944 0.953 1674528384 256 0.912 0.912 21859108 257 0.872 0.87812589871 258 0.697 0.716 28731108 259 0.699 0.693 23421302 260 0.6941.297 7870457 261 0.692 1.289 8126754

TABLE 8.5 R1-proton-NMR-peaklist FREQUENCY # ADDRESS [Hz] [PPM]INTENSITY 1 13957.6 3913.934 6.5215 20.81 2 15614.0 3550.810 5.9164 7.033 15656.8 3541.408 5.9008 14.16 4 15699.7 3532.016 5.8851 7.43 5 15839.63501.350 5.8340 2.56 6 15872.2 3494.204 5.8221 6.47 7 15905.1 3486.9925.8101 6.47 8 15932.8 3480.916 5.8000 2.49 9 17152.3 3213.554 5.354512.90 10 17853.4 3059.848 5.0984 17.52 11 17888.8 3052.093 5.0855 18.0312 18074.9 3011.286 5.0175 16.80 13 18110.2 3003.544 5.0046 17.26 1418524.3 2912.751 4.8533 15.71 15 18558.4 2905.282 4.8408 20.27 1618597.3 2896.749 4.8266 12.17 17 18638.6 2887.709 4.8116 5.88 18 18667.22881.421 4.8011 6.29 19 18680.9 2878.427 4.7961 6.65 20 18709.4 2872.1874.7857 5.58 21 18857.7 2839.672 4.7315 13.15 22 18891.0 2832.356 4.719315.61 23 18902.5 2829.844 4.7151 13.51 24 18918.1 2826.431 4.7095 13.2525 18996.6 2809.208 4.6808 8.60 26 19038.5 2800.032 4.6655 9.32 2719136.8 2778.471 4.6296 7.89 28 19151.7 2775.208 4.6241 6.99 29 19179.02769.220 4.6141 8.39 30 19194.1 2765.907 4.6086 7.85 31 19474.8 2704.3694.5061 16.52 32 19515.8 2695.385 4.4911 14.33 33 19529.0 2692.503 4.486320.98 34 19797.2 2633.699 4.3883 10.45 35 19819.7 2628.754 4.3801 17.6436 19850.7 2621.968 4.3688 26.32 37 19872.3 2617.228 4.3609 27.93 3819897.2 2611.771 4.3518 17.82 39 19930.6 2604.438 4.3396 11.93 4019963.7 2597.191 4.3275 8.30 41 20006.6 2587.791 4.3118 14.07 42 20031.72582.289 4.3027 11.75 43 20048.8 2578.545 4.2964 8.57 44 20076.52572.467 4.2863 11.71 45 20107.9 2565.584 4.2748 7.33 46 20151.62556.001 4.2589 14.11 47 20174.3 2551.024 4.2506 6.12 48 20196.32546.196 4.2425 13.56 49 20214.9 2542.128 4.2358 16.39 50 20247.52534.968 4.2238 26.96 51 20285.9 2526.555 4.2098 39.00 52 20305.42522.271 4.2027 24.70 53 20319.5 2519.190 4.1975 23.22 54 20346.42513.298 4.1877 7.91 55 20358.9 2510.546 4.1831 7.69 56 20379.8 2505.9594.1755 8.17 57 20422.0 2496.719 4.1601 10.22 58 20461.6 2488.043 4.14565.87 59 20487.5 2482.352 4.1362 7.27 60 20514.7 2476.402 4.1262 7.42 6120558.8 2466.727 4.1101 7.60 62 20596.2 2458.528 4.0965 31.95 63 20697.72436.266 4.0594 8.35 64 20735.2 2428.056 4.0457 13.12 65 20773.12419.740 4.0318 13.77 66 20811.2 2411.385 4.0179 12.76 67 20848.02403.322 4.0045 7.29 68 20890.7 2393.971 3.9889 7.87 69 20932.7 2384.7493.9735 10.17 70 20970.3 2376.499 3.9598 12.68 71 21017.7 2366.108 3.94256.44 72 21123.5 2342.921 3.9038 16.37 73 21559.5 2247.327 3.7445 11.7274 21604.4 2237.496 3.7282 11.07 75 22810.2 1973.141 3.2877 6.60 7622829.6 1968.890 3.2806 6.56 77 22863.1 1961.542 3.2684 6.70 78 22883.01957.180 3.2611 6.59 79 24645.4 1570.792 2.6173 4.82 80 24691.2 1560.7562.6006 5.30 81 25359.9 1414.147 2.3563 5.53 82 25407.3 1403.761 2.339010.56 83 25464.1 1391.301 2.3182 6.50 84 25529.5 1376.964 2.2943 2.87 8525976.1 1279.056 2.1312 4.43 86 26038.8 1265.317 2.1083 9.07 87 26101.11251.649 2.0855 4.68 88 26348.4 1197.433 1.9952 2.18 89 26423.1 1181.0671.9679 6.86 90 26495.7 1165.147 1.9414 40.57 91 26523.1 1159.138 1.931435.58 92 26528.4 1157.984 1.9295 36.42 93 26565.0 1149.953 1.9161 14.1494 26612.0 1139.641 1.8989 12.71 95 26673.0 1126.283 1.8766 9.66 9626829.5 1091.957 1.8194 48.60 97 26857.0 1085.929 1.8094 113.02 9827349.4 977.978 1.6295 11.86 99 27403.6 966.099 1.6097 12.07 100 27963.7843.313 1.4051 22.40 101 28018.6 831.275 1.3851 8.46 102 28254.5 779.5461.2989 11.85 103 28298.3 769.948 1.2829 12.55 104 28317.6 765.717 1.275913.04 105 28356.2 757.269 1.2618 86.45 106 28387.8 750.328 1.2502 81.24107 28464.0 733.633 1.2224 88.26 108 28533.4 718.414 1.1970 73.80 10928677.9 686.739 1.1443 3.89 110 28745.9 671.820 1.1194 3.59 111 28867.8645.098 1.0749 74.04 112 28987.7 618.804 1.0311 4.13 113 29051.5 604.8261.0078 8.69 114 29106.8 592.703 0.9876 4.79 115 29207.1 570.711 0.9509114.09 116 29320.8 545.779 0.9094 3.45 117 29410.0 526.225 0.8768 1.79118 29492.8 508.068 0.8466 1.41 119 29857.9 428.030 0.7132 9.46 12029911.7 416.233 0.6935 9.30

TABLE 8.6 13C NMR of R1 peak list DU = C:/Bruker/XWIN-NMR, USER = guest,NAME = chan, EXPNO = 30, PROCNO = 1176.659 ppm, F2 = 6.843 ppm, MI =0.06 cm, MAXI = 10000.00 cm, PC = 1.400 FREQUENCY # ADDRESS [Hz] [PPM]INTENSITY 1 5310.9 25331.195 167.8559 0.12 2 8227.3 22634.219 149.98451.57 3 8251.8 22611.535 149.8342 11.79 4 8281.1 22584.428 149.6546 12.505 8310.4 22557.355 149.4752 12.30 6 9381.7 21566.594 142.9100 0.11 710403.1 20622.062 136.6511 0.14 8 10577.3 20460.975 135.5836 1.08 910596.8 20442.969 135.4643 5.78 10 10623.6 20418.133 135.2998 6.16 1110650.4 20393.346 135.1355 6.01 12 11696.9 19425.617 128.7229 0.14 1312537.9 18647.846 123.5690 2.11 14 12557.4 18629.844 123.4497 10.62 1512584.3 18604.896 123.2844 11.25 16 12611.3 18580.010 123.1195 10.98 1713444.2 17809.705 118.0151 0.11 18 14249.2 17065.322 113.0825 0.15 1915319.0 16075.968 106.5266 0.21 20 15492.3 15915.713 105.4647 0.24 2115509.0 15900.229 105.3621 0.25 22 15823.2 15609.646 103.4365 0.18 2316269.0 15197.378 100.7047 0.16 24 18158.1 13450.413 89.1285 0.12 2519683.7 12039.604 79.7798 0.13 26 19797.3 11934.502 79.0834 0.14 2719929.2 11812.533 78.2752 0.23 28 19945.5 11797.527 78.1757 0.42 2920197.3 11564.589 76.6322 0.15 30 20211.9 11551.142 76.5431 0.16 3120226.2 11537.928 76.4555 0.14 32 20335.9 11436.434 75.7830 0.10 3320410.2 11367.753 75.3279 0.13 34 20473.6 11309.069 74.9390 0.38 3520617.2 11176.295 74.0592 0.13 36 20755.1 11048.805 73.2144 0.15 3720910.0 10905.522 72.2649 0.21 38 21019.3 10804.420 71.5950 0.15 3921047.5 10778.391 71.4225 0.37 40 21283.2 10560.453 69.9783 0.12 4121320.7 10525.739 69.7483 0.12 42 21375.2 10475.339 69.4143 0.14 4321430.8 10423.882 69.0733 0.13 44 22496.2 9438.663 62.5448 0.19 4522510.2 9425.676 62.4588 0.20 46 23646.3 8375.039 55.4968 0.13 4724915.8 7201.028 47.7173 0.13 48 25152.5 6982.204 46.2672 0.11 4925671.3 6502.366 43.0876 0.12 50 25868.3 6320.202 41.8805 0.10 5125921.2 6271.273 41.5563 0.11 52 26160.1 6050.418 40.0928 0.10 5326300.3 5920.724 39.2334 0.13 54 26396.6 5831.667 38.6433 0.10 5526722.2 5530.549 36.6479 0.11 56 26820.5 5439.679 36.0458 0.13 5727398.1 4905.475 32.5059 0.10 58 27774.8 4557.162 30.1978 0.23 5928148.7 4211.391 27.9066 0.23 60 28389.5 3988.645 26.4306 0.10 6128448.2 3934.385 26.0710 0.17 62 28512.9 3874.571 25.6747 0.10 6328828.2 3583.030 23.7428 0.11 64 29348.8 3101.522 20.5521 0.23 6529529.3 2934.591 19.4459 0.22 66 29650.2 2822.798 18.7051 0.26 6729719.5 2758.712 18.2805 0.11 68 29766.2 2715.508 17.9942 0.10 6929970.4 2526.664 16.7428 0.24 70 29985.5 2512.765 16.6507 0.17 7130140.7 2369.192 15.6993 0.22

TABLE 9.2 2D NMR (HMQC) chemical shift list of O54. Assignment w1 w2Data Height 1 123.540 5.337 10801059 2 106.679 4.875 7546160 3 106.7014.860 8527622 4 105.189 5.129 11004760 5 105.382 4.997 11249292 6105.173 5.114 7915383 7 103.375 4.725 9431190 8 100.663 6.513 18941426 988.873 3.329 5975702 10 88.850 3.307 5246604 11 79.767 4.210 7199634 1279.708 4.184 2885672 13 78.203 4.213 23826162 14 78.196 3.907 1378622815 76.818 4.148 6859174 16 76.490 3.944 5773632 17 76.642 3.749 751580818 76.628 3.730 8437212 19 75.842 4.089 22251034 20 75.318 4.241 265403221 75.330 4.213 9853961 22 75.103 4.299 7580958 23 75.169 4.279 637627224 74.986 4.025 11823145 25 75.168 4.003 10800605 26 74.084 4.3304901724 27 74.098 4.309 7830222 28 74.228 4.291 3975150 29 72.281 4.71117310028 30 72.230 4.625 8709239 31 72.206 4.606 7361591 32 71.479 4.22014407576 33 71.481 4.208 15081282 34 71.581 4.169 4582404 35 71.6134.150 8419769 36 71.628 4.121 5804582 37 71.637 4.099 7950110 38 71.6674.080 3119152 39 70.305 4.890 7622650 40 70.298 4.868 7628303 41 70.2654.314 4762902 42 69.983 4.676 6227999 43 69.918 4.649 6037791 44 70.1054.341 9963877 45 69.129 4.783 4704838 46 62.554 4.502 14998714 47 62.5324.477 14154374 48 62.548 4.362 11342701 49 62.537 4.344 8091356 5056.159 3.697 1838600 51 56.081 3.758 2144782 52 55.778 3.686 1783080 5355.590 0.717 5572202 54 55.588 0.694 6671325 55 47.732 1.608 4217994 5647.785 1.595 3965686 57 46.238 2.131 1734693 58 46.271 2.105 4919338 5946.299 2.072 2490009 60 46.279 1.304 4826865 61 46.276 1.272 4573323 6241.612 2.605 4436446 63 41.597 2.572 4591152 64 38.742 1.643 4256302 6538.730 1.612 3985649 66 38.641 1.031 3538408 67 32.508 1.413 10863677 6830.189 1.220 58134804 69 27.984 1.242 55524444 70 26.495 2.420 311303271 26.468 2.392 3183732 72 25.878 1.917 3435953 73 25.880 1.880 197994674 26.066 1.242 63345236 75 25.715 1.212 5054462 76 23.814 1.988 210403677 23.705 1.959 3237644 78 23.659 1.900 2754050 79 23.695 1.865 310809180 19.416 1.257 40949520 81 18.692 1.802 30867330 82 18.344 1.3893540974 83 18.308 1.413 6204910 84 17.911 2.330 3260134 85 17.942 2.3084212457 86 17.914 1.936 2468661 87 17.943 1.904 3914254 88 16.825 0.98048392540 89 16.640 1.072 52924908 90 15.701 0.947 60375744

TABLE 9.3 2D NMR (HMBC) chemical shift list of O54. DU =C:/Bruker/XWIN-NMR, USER = guest, NAME = chan, EXPNO = 37, PROCNO = 1F1PLO = 149.426 ppm, F1PHI = 11.307 ppm, F2PLO = 6.861 ppm, F2PHI =0.388 ppm MI = 2.00 cm, MAXI = 10000.00 cm, PC = 3.000 ADDRESS FREQUENCYrow [Hz]F1 [PPM]F1 # col [Hz]F2 [PPM]F2 INTENSITY 1 294 21551.752142.8145 728 1553.288 2.5881 3.15 2 294 21551.752 142.8145 791 1185.3451.9750 3.03 3 294 21551.752 142.8145 801 1127.853 1.8793 3.00 4 29421551.752 142.8145 866 744.836 1.2411 41.49 5 392 18639.387 123.5154 7281554.313 2.5898 2.70 6 392 18639.387 123.5154 730 1540.225 2.5664 2.11 7392 18639.387 123.5154 794 1164.901 1.9410 2.94 8 392 18639.387 123.5154801 1127.069 1.8779 3.61 9 478 16106.854 106.7334 585 2391.723 3.985120.41 10 478 16106.854 106.7334 653 1992.507 3.3200 3.17 11 48515890.850 105.3020 494 2924.288 4.8725 3.34 12 485 15890.850 105.3020516 2797.792 4.6617 3.27 13 485 15890.850 105.3020 547 2614.903 4.35705.99 14 485 15890.850 105.3020 583 2405.277 4.0077 11.83 15 49515600.868 103.3804 560 2539.046 4.2306 4.55 16 495 15600.868 103.3804574 2456.564 4.0932 3.63 17 509 15198.148 100.7118 312 3996.510 6.659111.15 18 509 15198.148 100.7118 341 3824.267 6.3721 12.11 19 50915198.148 100.7118 560 2539.421 4.2312 2.82 20 570 13411.609 88.8731 4952921.818 4.8684 18.81 21 570 13411.609 88.8731 797 1147.142 1.9114 2.7322 570 13411.609 88.8731 827 975.260 1.6250 6.25 23 570 13411.60988.8731 866 746.125 1.2432 69.46 24 570 13411.609 88.8731 892 589.8460.9828 48.36 25 616 12031.100 79.7251 561 2533.072 4.2207 10.23 26 61612031.100 79.7251 568 2492.076 4.1524 12.78 27 624 11792.174 78.1418 4693075.630 5.1247 3.71 28 624 11792.174 78.1418 481 3002.844 5.0034 2.5729 624 11792.174 78.1418 534 2690.732 4.4834 2.54 30 624 11792.17478.1418 547 2617.207 4.3608 2.56 31 624 11792.174 78.1418 561 2532.7654.2201 29.07 32 624 11792.174 78.1418 573 2460.286 4.0994 15.44 33 62411792.174 78.1418 580 2419.846 4.0320 13.41 34 624 11792.174 78.1418 5852390.343 3.9828 8.30 35 624 11792.174 78.1418 868 732.442 1.2204 3.23 36632 11558.919 76.5961 553 2577.697 4.2950 11.58 37 632 11558.919 76.5961563 2521.889 4.2020 3.02 38 632 11558.919 76.5961 573 2462.246 4.102613.81 39 632 11558.919 76.5961 861 772.953 1.2879 9.11 40 632 11558.91976.5961 864 755.650 1.2591 33.06 41 632 11558.919 76.5961 868 732.1701.2200 53.66 42 639 11373.424 75.3669 326 3911.898 6.5181 5.00 43 63911373.424 75.3669 563 2523.630 4.2049 25.40 44 639 11373.424 75.3669 5742456.045 4.0923 5.16 45 639 11373.424 75.3669 611 2240.865 3.7338 5.5746 639 11373.424 75.3669 808 1082.299 1.8034 2.40 47 639 11373.42475.3669 864 756.608 1.2607 2.78 48 639 11373.424 75.3669 868 732.3701.2203 5.47 49 646 11168.377 74.0082 511 2828.344 4.7127 8.44 50 64611168.377 74.0082 520 2772.974 4.6204 6.66 51 646 11168.377 74.0082 5632524.284 4.2060 5.35 52 646 11168.377 74.0082 808 1082.395 1.8035 49.6953 655 10899.106 72.2238 326 3911.679 6.5177 25.27 54 655 10899.10672.2238 498 2902.662 4.8365 2.42 55 655 10899.106 72.2238 511 2829.4144.7144 5.76 56 655 10899.106 72.2238 523 2756.376 4.5927 2.33 57 65510899.106 72.2238 532 2703.575 4.5048 2.40 58 655 10899.106 72.2238 5522588.313 4.3127 9.99 59 655 10899.106 72.2238 563 2523.240 4.2043 7.3560 655 10899.106 72.2238 808 1083.933 1.8061 2.26 61 659 10785.63771.4719 326 3912.133 6.5185 2.28 62 659 10785.637 71.4719 494 2925.9994.8754 2.36 63 659 10785.637 71.4719 516 2800.177 4.6657 3.39 64 65910785.637 71.4719 532 2702.493 4.5030 7.49 65 659 10785.637 71.4719 5342691.698 4.4850 7.86 66 659 10785.637 71.4719 547 2614.980 4.3571 2.3567 659 10785.637 71.4719 562 2525.201 4.2075 46.85 68 659 10785.63771.4719 584 2398.095 3.9958 2.02 69 665 10604.599 70.2723 326 3911.9566.5182 2.12 70 665 10604.599 70.2723 469 3075.252 5.1241 18.68 71 66510604.599 70.2723 481 3002.048 5.0021 3.29 72 665 10604.599 70.2723 5622525.138 4.2074 3.03 73 665 10604.599 70.2723 573 2460.794 4.1002 6.4674 665 10604.599 70.2723 808 1081.934 1.8027 3.10 75 666 10552.99069.9303 326 3912.459 6.5190 2.10 76 666 10552.990 69.9303 468 3077.1875.1273 13.23 77 666 10552.990 69.9303 481 3002.402 5.0027 10.72 78 66610552.990 69.9303 563 2522.129 4.2024 3.33 79 666 10552.990 69.9303 5732461.269 4.1010 4.47 80 666 10552.990 69.9303 808 1081.723 1.8024 2.7281 671 10429.136 69.1095 326 3911.639 6.5177 38.49 82 671 10429.13669.1095 552 2589.015 4.3139 8.97 83 671 10429.136 69.1095 808 1081.9871.8028 63.77 84 705 9425.537 62.4591 560 2542.206 4.2359 3.27 85 7059425.537 62.4591 562 2526.304 4.2094 2.08 86 740 8376.884 55.5101 827971.239 1.6183 3.45 87 740 8376.884 55.5101 849 843.561 1.4056 3.43 88740 8376.884 55.5101 866 746.292 1.2435 48.00 89 740 8376.884 55.5101892 590.131 0.9833 31.95 90 740 8376.884 55.5101 896 567.050 0.944825.60 91 780 7196.908 47.6909 447 3202.461 5.3360 4.85 92 780 7196.90847.6909 864 756.127 1.2599 2.83 93 780 7196.908 47.6909 868 731.6581.2191 3.78 94 780 7196.908 47.6909 883 642.229 1.0701 28.25 95 7807196.908 47.6909 896 567.166 0.9450 32.38 96 780 7196.908 47.6909 922414.895 0.6913 2.37 97 788 6979.996 46.2535 730 1540.055 2.5661 2.37 98788 6979.996 46.2535 864 755.679 1.2591 58.65 99 788 6979.996 46.2535868 731.892 1.2195 85.12 100 804 6505.572 43.1097 553 2577.654 4.29494.16 101 804 6505.572 43.1097 574 2455.343 4.0911 4.70 102 804 6505.57243.1097 755 1393.963 2.3227 3.13 103 804 6505.572 43.1097 858 789.6031.3157 3.35 104 804 6505.572 43.1097 861 773.435 1.2887 9.90 105 8046505.572 43.1097 867 737.064 1.2281 9.41 106 804 6505.572 43.1097 883642.755 1.0710 2.74 107 804 6505.572 43.1097 892 589.037 0.9815 2.09 108810 6311.923 41.8265 447 3202.295 5.3357 8.24 109 810 6311.923 41.8265755 1393.660 2.3221 6.43 110 810 6311.923 41.8265 830 955.929 1.59282.54 111 810 6311.923 41.8265 866 744.369 1.2403 45.72 112 810 6311.92341.8265 869 724.741 1.2076 3.80 113 810 6311.923 41.8265 883 642.2711.0702 53.84 114 810 6311.923 41.8265 892 589.734 0.9826 2.36 115 8146190.622 41.0227 447 3204.169 5.3389 2.27 116 814 6190.622 41.0227 866744.967 1.2413 4.20 117 814 6190.622 41.0227 883 641.291 1.0685 13.51118 814 6190.622 41.0227 892 590.266 0.9835 3.54 119 817 6122.21240.5694 849 842.541 1.4039 2.02 120 817 6122.212 40.5694 866 744.0981.2398 10.92 121 817 6122.212 40.5694 883 641.844 1.0695 19.65 122 8176122.212 40.5694 892 590.010 0.9831 4.84 123 819 6054.448 40.1203 8001128.653 1.8806 2.01 124 819 6054.448 40.1203 830 955.874 1.5927 5.24125 819 6054.448 40.1203 849 842.438 1.4037 7.20 126 819 6054.44840.1203 866 744.338 1.2402 39.00 127 819 6054.448 40.1203 883 642.0471.0698 49.93 128 819 6054.448 40.1203 892 589.724 0.9826 6.64 129 8196054.448 40.1203 896 566.654 0.9442 2.44 130 823 5927.002 39.2758 866746.066 1.2431 48.54 131 823 5927.002 39.2758 883 642.112 1.0699 7.22132 823 5927.002 39.2758 892 589.816 0.9828 60.87 133 823 5927.00239.2758 896 566.631 0.9441 5.86 134 823 5927.002 39.2758 922 415.4730.6923 7.24 135 827 5833.933 38.6590 827 970.335 1.6168 3.11 136 8275833.933 38.6590 865 746.825 1.2444 10.44 137 827 5833.933 38.6590 883642.035 1.0698 3.42 138 827 5833.933 38.6590 892 588.957 0.9813 11.20139 827 5833.933 38.6590 896 567.124 0.9450 55.77 140 830 5737.57038.0205 864 753.968 1.2563 3.79 141 830 5737.570 38.0205 868 731.4831.2188 4.47 142 830 5737.570 38.0205 883 641.875 1.0695 2.21 143 8305737.570 38.0205 892 589.272 0.9819 4.70 144 830 5737.570 38.0205 896567.206 0.9451 11.09 145 833 5652.819 37.4589 865 751.942 1.2529 5.17146 833 5652.819 37.4589 868 732.193 1.2200 5.78 147 833 5652.81937.4589 883 642.313 1.0702 2.64 148 833 5652.819 37.4589 892 590.1890.9834 2.92 149 833 5652.819 37.4589 896 567.196 0.9451 11.22 150 8375532.757 36.6633 747 1441.047 2.4011 2.33 151 837 5532.757 36.6633 830956.266 1.5934 6.70 152 837 5532.757 36.6633 849 840.791 1.4009 7.18 153837 5532.757 36.6633 864 754.417 1.2570 10.60 154 837 5532.757 36.6633868 733.768 1.2226 10.09 155 837 5532.757 36.6633 883 642.220 1.07012.62 156 837 5532.757 36.6633 896 567.041 0.9448 59.49 157 837 5532.75736.6633 922 415.510 0.6923 7.27 158 840 5447.502 36.0983 611 2241.4693.7348 3.44 159 840 5447.502 36.0983 775 1277.736 2.1290 2.18 160 8405447.502 36.0983 778 1261.898 2.1026 5.77 161 840 5447.502 36.0983 7801246.496 2.0769 2.77 162 840 5447.502 36.0983 861 771.393 1.2853 7.30163 840 5447.502 36.0983 864 755.513 1.2589 85.50 164 840 5447.50236.0983 868 731.684 1.2191 94.68 165 840 5447.502 36.0983 883 642.2111.0701 2.09 166 840 5447.502 36.0983 896 566.770 0.9444 11.25 167 8584901.029 32.4771 859 783.807 1.3060 2.31 168 858 4901.029 32.4771 883642.014 1.0697 38.68 169 858 4901.029 32.4771 922 416.874 0.6946 3.35170 870 4554.428 30.1803 611 2241.599 3.7350 9.79 171 870 4554.42830.1803 777 1263.228 2.1048 3.21 172 870 4554.428 30.1803 857 793.8841.3228 18.60 173 870 4554.428 30.1803 864 755.200 1.2583 93.90 174 8704554.428 30.1803 879 668.641 1.1141 19.16 175 870 4554.428 30.1803 892590.200 0.9834 3.11 176 881 4218.921 27.9570 855 807.262 1.3451 16.41177 881 4218.921 27.9570 864 755.803 1.2593 4.75 178 881 4218.92127.9570 876 683.031 1.1381 15.67 179 881 4218.921 27.9570 892 589.9180.9829 73.26 180 881 4218.921 27.9570 922 416.446 0.6939 2.87 181 8913935.921 26.0817 802 1120.176 1.8665 2.34 182 891 3935.921 26.0817 855806.480 1.3438 19.11 183 891 3935.921 26.0817 866 745.923 1.2429 8.92184 891 3935.921 26.0817 868 731.989 1.2197 5.17 185 891 3935.92126.0817 877 680.851 1.1344 18.56 186 891 3935.921 26.0817 892 589.6870.9825 4.65 187 893 3878.913 25.7039 791 1185.608 1.9755 2.05 188 8933878.913 25.7039 810 1072.605 1.7872 2.02 189 893 3878.913 25.7039 855805.970 1.3429 4.93 190 893 3878.913 25.7039 866 744.248 1.2401 44.75191 893 3878.913 25.7039 877 680.894 1.1345 4.45 192 893 3878.91325.7039 892 590.170 0.9834 3.96 193 925 2932.829 19.4346 611 2240.9543.7339 15.68 194 925 2932.829 19.4346 775 1278.753 2.1307 4.57 195 9252932.829 19.4346 778 1262.372 2.1034 11.84 196 925 2932.829 19.4346 7801246.392 2.0768 6.85 197 925 2932.829 19.4346 854 816.734 1.3609 22.66198 925 2932.829 19.4346 868 732.145 1.2199 93.22 199 925 2932.82919.4346 875 691.416 1.1521 21.54 200 933 2709.260 17.9531 553 2577.9184.2954 10.65 201 933 2709.260 17.9531 574 2456.969 4.0939 2.40 202 9332709.260 17.9531 728 1553.812 2.5890 2.47 203 933 2709.260 17.9531 7861214.060 2.0229 2.44 204 933 2709.260 17.9531 865 746.759 1.2443 4.64205 933 2709.260 17.9531 868 731.918 1.2195 5.50 206 938 2536.52016.8085 653 1992.054 3.3192 2.61 207 938 2536.520 16.8085 829 958.4761.5970 2.70 208 938 2536.520 16.8085 866 745.904 1.2428 52.11 209 9382536.520 16.8085 882 650.673 1.0842 17.18 210 938 2536.520 16.8085 895574.736 0.9576 3.28 211 938 2536.520 16.8085 903 526.041 0.8765 16.84212 938 2536.520 16.8085 922 416.280 0.6936 8.32 213 944 2375.48715.7414 830 955.713 1.5924 5.34 214 944 2375.487 15.7414 866 745.6841.2425 6.94 215 944 2375.487 15.7414 886 627.576 1.0457 21.31 216 9442375.487 15.7414 892 588.774 0.9810 3.00 217 944 2375.487 15.7414 907503.819 0.8395 21.33 218 944 2375.487 15.7414 922 415.374 0.6921 10.43

TABLE 9.5 Proton NMR peak list of O54. FREQUENCY # ADDRESS [Hz] [PPM]INTENSITY 1 7886.3 5225.509 8.7069 180.00 2 11038.4 4534.438 7.555472.73 3 12044.3 4313.911 7.1879 160.90 4 13878.4 3911.819 6.5180 14.53 517112.8 3202.719 5.3364 9.02 6 17674.0 3079.680 5.1314 10.52 7 17709.43071.938 5.1185 10.66 8 18004.6 3007.215 5.0107 10.57 9 18039.9 2999.4804.9978 10.78 10 18330.8 2935.701 4.8915 6.88 11 18377.3 2925.509 4.874516.06 12 18411.1 2918.099 4.8622 10.01 13 18591.4 2878.554 4.7963 3.5514 18621.6 2871.941 4.7853 4.53 15 18660.9 2863.325 4.7709 3.56 1618768.9 2839.657 4.7315 8.48 17 18801.9 2832.416 4.7194 15.95 18 18929.22804.496 4.6729 5.72 19 18974.3 2794.608 4.6564 6.90 20 19049.2 2778.1914.6291 5.41 21 19063.7 2775.007 4.6238 4.83 22 19091.3 2768.955 4.61375.79 23 19106.0 2765.737 4.6083 5.24 24 19395.7 2702.228 4.5025 11.91 2519447.9 2690.790 4.4835 16.03 26 19708.9 2633.561 4.3881 6.86 27 19731.82628.552 4.3798 8.37 28 19761.7 2621.989 4.3688 16.78 29 19784.12617.071 4.3606 18.07 30 19814.8 2610.350 4.3494 12.73 31 19834.72605.976 4.3421 13.55 32 19863.8 2599.612 4.3315 9.52 33 19909.82589.523 4.3147 13.96 34 19947.5 2581.249 4.3009 10.27 35 19990.62571.809 4.2852 7.86 36 20116.2 2544.275 4.2393 6.06 37 20155.1 2535.7504.2251 29.53 38 20188.0 2528.527 4.2131 36.53 39 20218.4 2521.859 4.202022.79 40 20258.5 2513.072 4.1873 10.10 41 20308.5 2502.111 4.1691 7.1742 20349.0 2493.228 4.1543 10.94 43 20384.7 2485.412 4.1412 7.98 4420454.4 2470.136 4.1158 8.00 45 20496.1 2460.979 4.1005 11.54 46 20522.92455.108 4.0908 20.70 47 20619.4 2433.953 4.0555 5.10 48 20655.02426.151 4.0425 7.46 49 20704.6 2415.271 4.0244 6.32 50 20741.8 2407.1194.0108 8.93 51 20763.7 2402.312 4.0028 6.78 52 20800.3 2394.290 3.98947.59 53 20840.0 2385.587 3.9749 4.23 54 20899.5 2372.534 3.9532 5.04 5520961.5 2358.953 3.9305 5.93 56 21011.8 2347.917 3.9122 8.88 57 21462.42249.135 3.7476 8.28 58 21507.4 2239.268 3.7311 7.60 59 22606.3 1998.3483.3297 4.57 60 22625.1 1994.242 3.3228 4.57 61 22658.9 1986.819 3.31054.74 62 22678.2 1982.598 3.3034 4.55 63 24608.6 1559.375 2.5983 3.63 6424657.0 1548.768 2.5806 3.98 65 25113.6 1448.658 2.4138 3.70 66 25160.11438.461 2.3968 4.03 67 25341.7 1398.662 2.3305 3.88 68 25394.7 1387.0362.3111 4.43 69 25900.2 1276.207 2.1264 3.15 70 25962.8 1262.484 2.10366.25 71 26025.4 1248.772 2.0807 3.23 72 26432.4 1159.530 1.9320 8.83 7326518.7 1140.610 1.9005 11.87 74 26686.8 1103.771 1.8391 2.00 75 26772.71084.927 1.8077 28.83 76 26800.2 1078.904 1.7977 28.10 77 27239.8982.535 1.6371 4.71 78 27287.4 972.099 1.6197 8.43 79 27366.6 954.7361.5908 4.35 80 27864.2 845.631 1.4090 14.94 81 28172.8 777.969 1.29639.42 82 28216.7 768.363 1.2803 9.50 83 28235.2 764.288 1.2735 9.84 8428276.7 755.206 1.2583 58.74 85 28321.2 745.447 1.2421 94.67 86 28380.5732.444 1.2204 58.53 87 28793.1 641.978 1.0697 50.29 88 28906.6 617.1141.0282 5.31 89 29031.6 589.696 0.9826 56.49 90 29134.1 567.228 0.945150.09 91 29765.3 428.852 0.7146 6.67 92 29818.8 417.128 0.6950 6.36DU = C: /Bruker/XWIN-NMR,USER = guest,NAME = chan,EXPNO = 35,PROCNO = 1F1 = 10.000 ppm,F2 = 0.000 ppm,MI = 2.00 cm,MAXI = 10000.00 cm,PC = 1.000

REFERENCES

-   (1) Chen, Q. 1995. Methods of study on pharmacology of Chinese    medicines. Press of People's Public Health, Beijing. p 892.-   (2) Huang, Zh. Sh., Liu, M. P., Chen, Ch. Zh. 1997. Study on effects    of Yangshou Dan on improving learning and retention. Chinese Journal    of combination of Chinese and west medicine, 9(17): 553.-   (3) Zhang, Y., Zhang, H. Y., Li, W. P. 1995. Study on effects of    Anjifu on improving intelligence, Chinese Bulletin of Pharmacology,    11(3): 233.-   (4) Yang, J., Wang, J., Feng, P. A. 2000. Study on effects of    Naokkangtai capsule on improving learning and retention in mice, New    Chinese Medicine and Clinical Pharmacology, 1(11): 29.-   (5) Yang, J., Wang, J., Zhang, J. Ch. 2000. Study on effects of    Crude saponins of peonies on improving learning and retention in    mice, Chinese journal of Pharmacology, 2(16): 46.-   (6) Xia, W. J., Jin, M. W., Zhang, L. 2000. Study on treatment of    senile dementia caused by angio-aging with Didang tang, Pharmacology    and Clinical of Chinese Medicines, 16 (4).-   (7) Bian, H. M., Yu, J. Z., Gong, J. N. 2000. Study on effects of    Tongmai Yizhi capsule on improving learning and retention in mice,    Pharmacology and Clinical of Chinese Medicines, 16 (5): 40.-   (8) Wei, X. L., Zhang, Y. X. 2000. Study of animal model for    studying senile dementia, Chinese journal of Pharmacology, 8(16):    372.-   (9) Bureau of Medicinal Police, Department of Public Health. Guide    line for study of effect of medicines for treatment of nervous    system diseases, in Guidebook of study of new medicine. p 45.-   (10) Zhang, D. Sh., Zhang, J. T. 2000. Effects of crude Ginseng    saponins on improving impairment induced by B-peptide, Chinese    journal of Pharmacology, 8(16): 22.

1-170. (canceled)
 171. A compound comprising the following structure:[STRUCTURE Y];

a chemical named 3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21,22-O-diangeloyl-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene;a salt thereof; or a derivative thereof.
 172. The compound of claim 171,wherein the structure is synthesized.
 173. The compound of claim 171,wherein the structure is isolated from natural sources.
 174. A compoundcomprising the following structure: [STRUCTURE Y1];

a chemical named3-O-[β-D-galactopyranosyl(1→2)]-α-L-arabinofuranosyl(1→3)-β-D-glucuronopyranosyl-21-O-(3,4-diangeloyl)-α-L-rhamnophyranosyl-22-O-acetyl-3β,16α,21β,22α,28-pentahydroxyolean-12-ene;a salt thereof; or a derivative thereof.
 175. The compound of claim 174,wherein the structure is synthesized.
 176. The compound of claim 174,wherein the structure is isolated from natural sources.
 177. Acomposition for regulating cell growth, comprising an effective amountof the compound of claim
 171. 178. A composition for regulating cellgrowth, comprising an effective amount of the compound of claim 174.179. A composition for inhibiting tumor cell growth, comprising aneffective amount of the compound of claim
 171. 180. A composition forinhibiting tumor cell growth, comprising an effective amount of thecompound of claim
 174. 181. The composition of claim 179, where thetumor is ovarian cancer, bladder cancer, prostate cancer, bone cancer orleukemia.
 182. The composition of claim 180, where the tumor is ovariancancer, bladder cancer, prostate cancer, bone cancer or leukemia.
 183. Afood for inhibiting tumor growth, comprising an effective amount of thecomposition of claim
 179. 184. A food for inhibiting tumor growth,comprising an effective amount of the composition of claim
 180. 185. Apharmaceutical composition for inhibiting tumor cell growth, comprisingan effective amount of the compound of claim 171 in a pharmaceuticallysuitable carrier.
 186. A pharmaceutical composition for inhibiting tumorcell growth, comprising an effective amount of the compound of claim 174in a pharmaceutically suitable carrier.